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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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Reducing of HIV-1 replication by lentivirus-mediated expression of Hutat2:Fc in primary hMDM.(A) Kinetics of HIV-1Ba−L replications (HIV-1 p24 levels). The data showed a significant reduction of HIV-1 replication in both the TD-hMDM and Hutat2:Fc culture groups as compared to hMDM (P <0.01), but no statistical difference among TD-hMDM, Hutat2:Fc, and Anti-Tat groups (P >0.05). (B) Lentiviral vectors HR-Hutat2 transduction suppresses HIV-1 cytopathicity and the expression of p24 in hMDM cultures. Normal hMDM and HR-Hutat2 transduced hMDM were exposed to HIV-1Ba-L, and examined before and on day 24 post-viral infection using a 10× objective. It can be readily appreciated that either HR-Hutat2 transduction or Hutat2:Fc strongly suppressed HIV-1-mediated cytopathic effects, resulting in a reduction in the number of giant cells in the culture. In addition, HIV-1 p24 immunofluorescent staining showed that HR-Hutat2 transduction and Hutat2:Fc reduced the expression of HIV-1 p24 intracellularly. Images were acquired as described in Figure 1. hMDM, Normal hMDM; TD-hMDM, HR-Hutat2 transduced hMDM; Anti-Tat, Non-transduced hMDM treated with anti-HIV-1 Tat antibody; Hutat2,Fc, Normal hMDM treated with conditioned medium from HR-Hutat2 transduced hMDM; D24 post-infection, Day 24 post-HIV-1-infection; p24, HIV-1 p24 immunofluorescent staining; White arrow, HIV-1-induced cytopathic effect. The blood of three donors was used in this assay. Results represent mean values from triple independent experiments and error bars denote the s.e.m. Scale bar = 100 μm.
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Fig5: Reducing of HIV-1 replication by lentivirus-mediated expression of Hutat2:Fc in primary hMDM.(A) Kinetics of HIV-1Ba−L replications (HIV-1 p24 levels). The data showed a significant reduction of HIV-1 replication in both the TD-hMDM and Hutat2:Fc culture groups as compared to hMDM (P <0.01), but no statistical difference among TD-hMDM, Hutat2:Fc, and Anti-Tat groups (P >0.05). (B) Lentiviral vectors HR-Hutat2 transduction suppresses HIV-1 cytopathicity and the expression of p24 in hMDM cultures. Normal hMDM and HR-Hutat2 transduced hMDM were exposed to HIV-1Ba-L, and examined before and on day 24 post-viral infection using a 10× objective. It can be readily appreciated that either HR-Hutat2 transduction or Hutat2:Fc strongly suppressed HIV-1-mediated cytopathic effects, resulting in a reduction in the number of giant cells in the culture. In addition, HIV-1 p24 immunofluorescent staining showed that HR-Hutat2 transduction and Hutat2:Fc reduced the expression of HIV-1 p24 intracellularly. Images were acquired as described in Figure 1. hMDM, Normal hMDM; TD-hMDM, HR-Hutat2 transduced hMDM; Anti-Tat, Non-transduced hMDM treated with anti-HIV-1 Tat antibody; Hutat2,Fc, Normal hMDM treated with conditioned medium from HR-Hutat2 transduced hMDM; D24 post-infection, Day 24 post-HIV-1-infection; p24, HIV-1 p24 immunofluorescent staining; White arrow, HIV-1-induced cytopathic effect. The blood of three donors was used in this assay. Results represent mean values from triple independent experiments and error bars denote the s.e.m. Scale bar = 100 μm.

Mentions: hMDM was transduced with HR-Hutat2 on DIV 7 and DIV 8 and cultured for 6 days, then normal hMDM, HR-Hutat2-transduced hMDM, and hMDM supplemented with anti-HIV-1 Tat or with the conditioned medium from HR-Hutat2-transduced hMDM were infected with HIV-1Ba-L, respectively. The level of HIV-1 p24 production in these cultures was quantified by an ELISA assay (Figure 5A). HIV-1Ba-L replication (p24 level) was detected in the control hMDM shortly after virus inoculation (day 3) and gradually increased with post-infection time, reaching the peak level by day 18 post-infection. The level of viral production dramatically suppressed (by 9- to 16-fold) in transduced hMDM-Hutat2 and normal hMDM supplemented with hMDM-Hutat2-conditioned medium or with anti-HIV-1 Tat antibody as compared to normal hMDM cultures (Figure 5A). These results suggest that the lentiviral vector-mediated Hutat2:Fc gene transfer conferred a significant degree of protection against wild-type HIV-1 infection in primary hMDM (P <0.01). In addition, the secreted Hutat2:Fc from transduced hMDM can suppress HIV-1Ba-L propagation as an anti-HIV-1 Tat antibody. In agreement with this, an HIV-1-induced cytopathic effect in non-transduced hMDM was evident by the presence of abnormally large cells, multinucleated cells, and debris resulting from late stages of cell death. As a comparison, only very modest levels of HIV-1-induced cytopathic effects were observed in the transduced cultures or non-transduced culture supplemented with Hutat2:Fc conditioned medium (Figure 5B). Furthermore, although almost all of hMDM were infected by HIV-1Ba-L after a 24-day culture period, the fluorescent signals of p24 staining in transduced hMDM or in normal hMDM treated with hMDM-Hutat2 conditioned medium were much weaker as compared to hMDM control (Figure 5B; p24 panel). These findings illustrate that although Hutat2:Fc is unable to completely block the cells from infection by HIV, lentiviral vector HR-Hutat2-transduced hMDM (intracellular Hutat2:Fc) and the Hutat2:Fc secreted from vector-transduced hMDM (extracellular Hutat2:Fc) are able to suppress HIV-1 replication and the spread of viral infection in macrophages.Figure 5


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)

Reducing of HIV-1 replication by lentivirus-mediated expression of Hutat2:Fc in primary hMDM.(A) Kinetics of HIV-1Ba−L replications (HIV-1 p24 levels). The data showed a significant reduction of HIV-1 replication in both the TD-hMDM and Hutat2:Fc culture groups as compared to hMDM (P <0.01), but no statistical difference among TD-hMDM, Hutat2:Fc, and Anti-Tat groups (P >0.05). (B) Lentiviral vectors HR-Hutat2 transduction suppresses HIV-1 cytopathicity and the expression of p24 in hMDM cultures. Normal hMDM and HR-Hutat2 transduced hMDM were exposed to HIV-1Ba-L, and examined before and on day 24 post-viral infection using a 10× objective. It can be readily appreciated that either HR-Hutat2 transduction or Hutat2:Fc strongly suppressed HIV-1-mediated cytopathic effects, resulting in a reduction in the number of giant cells in the culture. In addition, HIV-1 p24 immunofluorescent staining showed that HR-Hutat2 transduction and Hutat2:Fc reduced the expression of HIV-1 p24 intracellularly. Images were acquired as described in Figure 1. hMDM, Normal hMDM; TD-hMDM, HR-Hutat2 transduced hMDM; Anti-Tat, Non-transduced hMDM treated with anti-HIV-1 Tat antibody; Hutat2,Fc, Normal hMDM treated with conditioned medium from HR-Hutat2 transduced hMDM; D24 post-infection, Day 24 post-HIV-1-infection; p24, HIV-1 p24 immunofluorescent staining; White arrow, HIV-1-induced cytopathic effect. The blood of three donors was used in this assay. Results represent mean values from triple independent experiments and error bars denote the s.e.m. Scale bar = 100 μm.
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Fig5: Reducing of HIV-1 replication by lentivirus-mediated expression of Hutat2:Fc in primary hMDM.(A) Kinetics of HIV-1Ba−L replications (HIV-1 p24 levels). The data showed a significant reduction of HIV-1 replication in both the TD-hMDM and Hutat2:Fc culture groups as compared to hMDM (P <0.01), but no statistical difference among TD-hMDM, Hutat2:Fc, and Anti-Tat groups (P >0.05). (B) Lentiviral vectors HR-Hutat2 transduction suppresses HIV-1 cytopathicity and the expression of p24 in hMDM cultures. Normal hMDM and HR-Hutat2 transduced hMDM were exposed to HIV-1Ba-L, and examined before and on day 24 post-viral infection using a 10× objective. It can be readily appreciated that either HR-Hutat2 transduction or Hutat2:Fc strongly suppressed HIV-1-mediated cytopathic effects, resulting in a reduction in the number of giant cells in the culture. In addition, HIV-1 p24 immunofluorescent staining showed that HR-Hutat2 transduction and Hutat2:Fc reduced the expression of HIV-1 p24 intracellularly. Images were acquired as described in Figure 1. hMDM, Normal hMDM; TD-hMDM, HR-Hutat2 transduced hMDM; Anti-Tat, Non-transduced hMDM treated with anti-HIV-1 Tat antibody; Hutat2,Fc, Normal hMDM treated with conditioned medium from HR-Hutat2 transduced hMDM; D24 post-infection, Day 24 post-HIV-1-infection; p24, HIV-1 p24 immunofluorescent staining; White arrow, HIV-1-induced cytopathic effect. The blood of three donors was used in this assay. Results represent mean values from triple independent experiments and error bars denote the s.e.m. Scale bar = 100 μm.
Mentions: hMDM was transduced with HR-Hutat2 on DIV 7 and DIV 8 and cultured for 6 days, then normal hMDM, HR-Hutat2-transduced hMDM, and hMDM supplemented with anti-HIV-1 Tat or with the conditioned medium from HR-Hutat2-transduced hMDM were infected with HIV-1Ba-L, respectively. The level of HIV-1 p24 production in these cultures was quantified by an ELISA assay (Figure 5A). HIV-1Ba-L replication (p24 level) was detected in the control hMDM shortly after virus inoculation (day 3) and gradually increased with post-infection time, reaching the peak level by day 18 post-infection. The level of viral production dramatically suppressed (by 9- to 16-fold) in transduced hMDM-Hutat2 and normal hMDM supplemented with hMDM-Hutat2-conditioned medium or with anti-HIV-1 Tat antibody as compared to normal hMDM cultures (Figure 5A). These results suggest that the lentiviral vector-mediated Hutat2:Fc gene transfer conferred a significant degree of protection against wild-type HIV-1 infection in primary hMDM (P <0.01). In addition, the secreted Hutat2:Fc from transduced hMDM can suppress HIV-1Ba-L propagation as an anti-HIV-1 Tat antibody. In agreement with this, an HIV-1-induced cytopathic effect in non-transduced hMDM was evident by the presence of abnormally large cells, multinucleated cells, and debris resulting from late stages of cell death. As a comparison, only very modest levels of HIV-1-induced cytopathic effects were observed in the transduced cultures or non-transduced culture supplemented with Hutat2:Fc conditioned medium (Figure 5B). Furthermore, although almost all of hMDM were infected by HIV-1Ba-L after a 24-day culture period, the fluorescent signals of p24 staining in transduced hMDM or in normal hMDM treated with hMDM-Hutat2 conditioned medium were much weaker as compared to hMDM control (Figure 5B; p24 panel). These findings illustrate that although Hutat2:Fc is unable to completely block the cells from infection by HIV, lentiviral vector HR-Hutat2-transduced hMDM (intracellular Hutat2:Fc) and the Hutat2:Fc secreted from vector-transduced hMDM (extracellular Hutat2:Fc) are able to suppress HIV-1 replication and the spread of viral infection in macrophages.Figure 5

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