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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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Relative gene expression levels of theHutat2:FcandEGFPgenes in transduced cells and quantification of Hutat2:Fc in conditioned mediums.(A) Detection of Hutat2 and EGFP mRNA in HR-Hutat2 transduced cells by a RT-PCR qualitative analysis. HTB-Hutat2, HR-Hutat2 transduced HTB-11 RNA; U937-Hutat2, HR-Hutat2 transduced U937 RNA; hMDM-Hutat2, HR-Hutat2 transduced hMDM RNA; U937, non-transduced U937 RNA; IC, Internal control RNA from K562 cell line; NTC, No template control; RTase (−), RTase negative control. (B and C) Quantitative real-time PCR analysis of Hutat2 and EGFP gene expression levels in transduced HTB-11 and U937 cells compared with that in transduced hMDM (*P <0.01). (D) Comparison of Hutat2:Fc secretion level between transduced HTB-11 and U937 within 24 hours (*P <0.01); 1 × 106 cells were plated into a T-75 flask and the mediums were collected 24 hours later. Hutat2:Fc was quantified by a human IgG ELISA method. (E and F) Detection of Hutat2:Fc proteins in cell lysate and supernatant of transduced cells by Western blotting. (G) Detection of stable secretion of Hutat2:Fc in conditioned mediums from HR-Hutat2 transduced HTB-11 (HTB-Hutat2) and U937 cells (U937-Hutat2). Cells were passaged totally 20 times and an ELISA assay was performed every fifth passage. (H and I) The accumulation of Hutat2:Fc in mediums from transduced HTB-11 and U937 cells; 1 × 106 cells were plated into a T-75 flask and the mediums were collected every 24 hours for 4 days. (J) Kinetics of Hutat2:Fc levels in cell culture supernatants of transduced hMDM at different MOI after transduction. The levels of secreted Hutat2:Fc were peak on day 9 post-transduction. The concentrations of Hutat2:Fc were higher at MOI 50 than at MOI 10 in mediums of transduced hMDM at each time point (P <0.01). Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.
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Fig2: Relative gene expression levels of theHutat2:FcandEGFPgenes in transduced cells and quantification of Hutat2:Fc in conditioned mediums.(A) Detection of Hutat2 and EGFP mRNA in HR-Hutat2 transduced cells by a RT-PCR qualitative analysis. HTB-Hutat2, HR-Hutat2 transduced HTB-11 RNA; U937-Hutat2, HR-Hutat2 transduced U937 RNA; hMDM-Hutat2, HR-Hutat2 transduced hMDM RNA; U937, non-transduced U937 RNA; IC, Internal control RNA from K562 cell line; NTC, No template control; RTase (−), RTase negative control. (B and C) Quantitative real-time PCR analysis of Hutat2 and EGFP gene expression levels in transduced HTB-11 and U937 cells compared with that in transduced hMDM (*P <0.01). (D) Comparison of Hutat2:Fc secretion level between transduced HTB-11 and U937 within 24 hours (*P <0.01); 1 × 106 cells were plated into a T-75 flask and the mediums were collected 24 hours later. Hutat2:Fc was quantified by a human IgG ELISA method. (E and F) Detection of Hutat2:Fc proteins in cell lysate and supernatant of transduced cells by Western blotting. (G) Detection of stable secretion of Hutat2:Fc in conditioned mediums from HR-Hutat2 transduced HTB-11 (HTB-Hutat2) and U937 cells (U937-Hutat2). Cells were passaged totally 20 times and an ELISA assay was performed every fifth passage. (H and I) The accumulation of Hutat2:Fc in mediums from transduced HTB-11 and U937 cells; 1 × 106 cells were plated into a T-75 flask and the mediums were collected every 24 hours for 4 days. (J) Kinetics of Hutat2:Fc levels in cell culture supernatants of transduced hMDM at different MOI after transduction. The levels of secreted Hutat2:Fc were peak on day 9 post-transduction. The concentrations of Hutat2:Fc were higher at MOI 50 than at MOI 10 in mediums of transduced hMDM at each time point (P <0.01). Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.

Mentions: Furthermore, the transcriptional profiling for the integrated Hutat2 and EGFP genes in transduced HTB-11, U937, and hMDM were examined by RT-PCR analysis (Figure 2A) and confirmed by a real-time PCR test. The expression of Hutat2 and EGFP genes in transduced cells was normalized with three reference genes (ACTB, GK, and Ezrin) and compared with transduced hMDM. The expression levels of the Hutat2 gene in transduced HTB-11 and transduced U937 were 162.5- and 9.0-fold higher than that in transduced hMDM, respectively, while the expression level of the Hutat2 gene in transduced HTB-11 was 18.1-fold higher than that in transduced U937 (Figure 2B). In addition, the expression levels of EGFP in transduced HTB-11 and U937 cells were 89.7- and 4.4-fold higher than that determined in transduced hMDM, respectively (Figure 2C). The difference in the gene expression between different transduced cells was further confirmed by an ELISA quantification of Hutat2:Fc secreted in the supernatants of transduced HTB-11 and U937 cells. It was shown that the secretion of Hutat2:Fc in the supernatants of transduced HTB-11 was 17.1-fold higher than that in the supernatants of transduced U937 cells (2.39 ± 0.11 μg/106 cells/24 h compared with 0.14 ± 0.04 μg/106 cells/24 h, P <0.01) (Figure 2D).Figure 2


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)

Relative gene expression levels of theHutat2:FcandEGFPgenes in transduced cells and quantification of Hutat2:Fc in conditioned mediums.(A) Detection of Hutat2 and EGFP mRNA in HR-Hutat2 transduced cells by a RT-PCR qualitative analysis. HTB-Hutat2, HR-Hutat2 transduced HTB-11 RNA; U937-Hutat2, HR-Hutat2 transduced U937 RNA; hMDM-Hutat2, HR-Hutat2 transduced hMDM RNA; U937, non-transduced U937 RNA; IC, Internal control RNA from K562 cell line; NTC, No template control; RTase (−), RTase negative control. (B and C) Quantitative real-time PCR analysis of Hutat2 and EGFP gene expression levels in transduced HTB-11 and U937 cells compared with that in transduced hMDM (*P <0.01). (D) Comparison of Hutat2:Fc secretion level between transduced HTB-11 and U937 within 24 hours (*P <0.01); 1 × 106 cells were plated into a T-75 flask and the mediums were collected 24 hours later. Hutat2:Fc was quantified by a human IgG ELISA method. (E and F) Detection of Hutat2:Fc proteins in cell lysate and supernatant of transduced cells by Western blotting. (G) Detection of stable secretion of Hutat2:Fc in conditioned mediums from HR-Hutat2 transduced HTB-11 (HTB-Hutat2) and U937 cells (U937-Hutat2). Cells were passaged totally 20 times and an ELISA assay was performed every fifth passage. (H and I) The accumulation of Hutat2:Fc in mediums from transduced HTB-11 and U937 cells; 1 × 106 cells were plated into a T-75 flask and the mediums were collected every 24 hours for 4 days. (J) Kinetics of Hutat2:Fc levels in cell culture supernatants of transduced hMDM at different MOI after transduction. The levels of secreted Hutat2:Fc were peak on day 9 post-transduction. The concentrations of Hutat2:Fc were higher at MOI 50 than at MOI 10 in mediums of transduced hMDM at each time point (P <0.01). Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.
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Fig2: Relative gene expression levels of theHutat2:FcandEGFPgenes in transduced cells and quantification of Hutat2:Fc in conditioned mediums.(A) Detection of Hutat2 and EGFP mRNA in HR-Hutat2 transduced cells by a RT-PCR qualitative analysis. HTB-Hutat2, HR-Hutat2 transduced HTB-11 RNA; U937-Hutat2, HR-Hutat2 transduced U937 RNA; hMDM-Hutat2, HR-Hutat2 transduced hMDM RNA; U937, non-transduced U937 RNA; IC, Internal control RNA from K562 cell line; NTC, No template control; RTase (−), RTase negative control. (B and C) Quantitative real-time PCR analysis of Hutat2 and EGFP gene expression levels in transduced HTB-11 and U937 cells compared with that in transduced hMDM (*P <0.01). (D) Comparison of Hutat2:Fc secretion level between transduced HTB-11 and U937 within 24 hours (*P <0.01); 1 × 106 cells were plated into a T-75 flask and the mediums were collected 24 hours later. Hutat2:Fc was quantified by a human IgG ELISA method. (E and F) Detection of Hutat2:Fc proteins in cell lysate and supernatant of transduced cells by Western blotting. (G) Detection of stable secretion of Hutat2:Fc in conditioned mediums from HR-Hutat2 transduced HTB-11 (HTB-Hutat2) and U937 cells (U937-Hutat2). Cells were passaged totally 20 times and an ELISA assay was performed every fifth passage. (H and I) The accumulation of Hutat2:Fc in mediums from transduced HTB-11 and U937 cells; 1 × 106 cells were plated into a T-75 flask and the mediums were collected every 24 hours for 4 days. (J) Kinetics of Hutat2:Fc levels in cell culture supernatants of transduced hMDM at different MOI after transduction. The levels of secreted Hutat2:Fc were peak on day 9 post-transduction. The concentrations of Hutat2:Fc were higher at MOI 50 than at MOI 10 in mediums of transduced hMDM at each time point (P <0.01). Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.
Mentions: Furthermore, the transcriptional profiling for the integrated Hutat2 and EGFP genes in transduced HTB-11, U937, and hMDM were examined by RT-PCR analysis (Figure 2A) and confirmed by a real-time PCR test. The expression of Hutat2 and EGFP genes in transduced cells was normalized with three reference genes (ACTB, GK, and Ezrin) and compared with transduced hMDM. The expression levels of the Hutat2 gene in transduced HTB-11 and transduced U937 were 162.5- and 9.0-fold higher than that in transduced hMDM, respectively, while the expression level of the Hutat2 gene in transduced HTB-11 was 18.1-fold higher than that in transduced U937 (Figure 2B). In addition, the expression levels of EGFP in transduced HTB-11 and U937 cells were 89.7- and 4.4-fold higher than that determined in transduced hMDM, respectively (Figure 2C). The difference in the gene expression between different transduced cells was further confirmed by an ELISA quantification of Hutat2:Fc secreted in the supernatants of transduced HTB-11 and U937 cells. It was shown that the secretion of Hutat2:Fc in the supernatants of transduced HTB-11 was 17.1-fold higher than that in the supernatants of transduced U937 cells (2.39 ± 0.11 μg/106 cells/24 h compared with 0.14 ± 0.04 μg/106 cells/24 h, P <0.01) (Figure 2D).Figure 2

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