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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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Protection of the conditioned medium containing Hutat2:Fc against HIV-1 Tat86-mediated neurotoxicity in primary mouse neurons. Mouse cortical neurons cultured in 24-well plates were treated with HIV-1 Tat86 (Clade B, 500 nM) alone, or Tat with conditioned mediums from HR-Hutat2-transduced hMDM or HTB-11 (1:5 dilution) on day 6 in vitro (DIV 6) for 3 days. Treatment with Tat plus anti-Tat monoclonal antibody was used as a positive control, while Tat plus the conditioned medium from HR-A3H5 transduced HTB-11 was used as a negative control, respectively. (A) Representative images of primary mouse cortical neurons which were treated with HIV-1 Tat86 or Tat86 plus the conditioned medium from HR-Hutat2-transduced hMDM. Cells were counterstained with anti-MAP2 (MAP2), FITC-dUTP (TUNEL), and DAPI (Nuclei). Images of MAP2, TUNEL, and Nuclei were merged together (Merge). The survived neurons were the cells which were positive for MAP2 and DAPI but negative for TUNEL staining. Tat, Neurons treated with HIV-1 Tat86 alone; Tat/hMDM-Hutat2 medium, Neurons treated with HIV-1 Tat86 plus the conditioned medium of transduced hMDM; Normal control, Untreated neurons. Images were acquired as described in Figure 1. (B) Comparison of relative rates of neuron survival after treatment. The neuron survival rate of untreated neurons was defined as 100%. The relative neuron survival rate was increased by about 10% by adding Hutat2:Fc containing medium from transduced hMDM (*P <0.05 vs. treatment with Tat alone). However, the rate was still lower than normal neurons, neurons treated with Tat86 plus HTB-Hutat2 medium, and Tat86 plus anti-Tat antibody (#P <0.01). Each value is the mean obtained from five random fields of three independent experiments using a 20× objective. Error bars denote the s.e.m. Scale bar = 100 μm.
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Fig4: Protection of the conditioned medium containing Hutat2:Fc against HIV-1 Tat86-mediated neurotoxicity in primary mouse neurons. Mouse cortical neurons cultured in 24-well plates were treated with HIV-1 Tat86 (Clade B, 500 nM) alone, or Tat with conditioned mediums from HR-Hutat2-transduced hMDM or HTB-11 (1:5 dilution) on day 6 in vitro (DIV 6) for 3 days. Treatment with Tat plus anti-Tat monoclonal antibody was used as a positive control, while Tat plus the conditioned medium from HR-A3H5 transduced HTB-11 was used as a negative control, respectively. (A) Representative images of primary mouse cortical neurons which were treated with HIV-1 Tat86 or Tat86 plus the conditioned medium from HR-Hutat2-transduced hMDM. Cells were counterstained with anti-MAP2 (MAP2), FITC-dUTP (TUNEL), and DAPI (Nuclei). Images of MAP2, TUNEL, and Nuclei were merged together (Merge). The survived neurons were the cells which were positive for MAP2 and DAPI but negative for TUNEL staining. Tat, Neurons treated with HIV-1 Tat86 alone; Tat/hMDM-Hutat2 medium, Neurons treated with HIV-1 Tat86 plus the conditioned medium of transduced hMDM; Normal control, Untreated neurons. Images were acquired as described in Figure 1. (B) Comparison of relative rates of neuron survival after treatment. The neuron survival rate of untreated neurons was defined as 100%. The relative neuron survival rate was increased by about 10% by adding Hutat2:Fc containing medium from transduced hMDM (*P <0.05 vs. treatment with Tat alone). However, the rate was still lower than normal neurons, neurons treated with Tat86 plus HTB-Hutat2 medium, and Tat86 plus anti-Tat antibody (#P <0.01). Each value is the mean obtained from five random fields of three independent experiments using a 20× objective. Error bars denote the s.e.m. Scale bar = 100 μm.

Mentions: In addition, these protective effects of Hutat2:Fc in the conditioned mediums were further evaluated using primary cultures of mouse neurons. Early postnatal (P0) Balb/c mouse neurons from cortex were isolated and cultured for 6 DIVs. The purity of the cultures were >95% neurons proved by MAP2 and glial fibrillary acidic protein immunocytochemistry staining (data not shown). The representative images of normal neurons and neurons treated with Tat86 or Tat86 plus Hutat2:Fc containing mediums from the transduced hMDM are shown in Figure 4A. Tat-treated mouse neurons showed increased numbers of cell apoptosis (Figure 4A TUNEL panel), loss of dendritic arbor, as well as a shorter dendrite length (Figure 4A; MAP2 panel). The relative rate of neuron survival was similar among normal neurons, neurons treated with Tat86 plus conditioned medium from HTB-Hutat2 (93.0 ± 4.5%), and neurons treated with Tat86 plus anti-Tat antibody (97.0 ± 7.2%). Compared with Tat exposure alone, the relative rate of neuron survival was increased by 10%, from 69.3 ± 8.9% to 79.4 ± 7.9% in the presence of conditioned medium from HR-Hutat2-transduced hMDM (P <0.05). However, the neuron survival rates were not significantly changed when adding HTB-A3H5 medium (66.6 ± 9.6% versus 69.3 ± 8.9%, P >0.05; Figure 4B). These results indicate that Hutat2:Fc released from transduced hMDM and HTB-11 could neutralize HIV-1 Tat86-induced neurotoxicity as an anti-Tat antibody in vitro, whereas A3H5:Fc released from HTB-A3H5 control does not have that biological effect. In comparison, the protective level of Hutat2:Fc from the conditioned medium of transduced hMDM was lower than that obtained from the use of transduced HTB-11 medium and the commercial anti-Tat antibody.Figure 4


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)

Protection of the conditioned medium containing Hutat2:Fc against HIV-1 Tat86-mediated neurotoxicity in primary mouse neurons. Mouse cortical neurons cultured in 24-well plates were treated with HIV-1 Tat86 (Clade B, 500 nM) alone, or Tat with conditioned mediums from HR-Hutat2-transduced hMDM or HTB-11 (1:5 dilution) on day 6 in vitro (DIV 6) for 3 days. Treatment with Tat plus anti-Tat monoclonal antibody was used as a positive control, while Tat plus the conditioned medium from HR-A3H5 transduced HTB-11 was used as a negative control, respectively. (A) Representative images of primary mouse cortical neurons which were treated with HIV-1 Tat86 or Tat86 plus the conditioned medium from HR-Hutat2-transduced hMDM. Cells were counterstained with anti-MAP2 (MAP2), FITC-dUTP (TUNEL), and DAPI (Nuclei). Images of MAP2, TUNEL, and Nuclei were merged together (Merge). The survived neurons were the cells which were positive for MAP2 and DAPI but negative for TUNEL staining. Tat, Neurons treated with HIV-1 Tat86 alone; Tat/hMDM-Hutat2 medium, Neurons treated with HIV-1 Tat86 plus the conditioned medium of transduced hMDM; Normal control, Untreated neurons. Images were acquired as described in Figure 1. (B) Comparison of relative rates of neuron survival after treatment. The neuron survival rate of untreated neurons was defined as 100%. The relative neuron survival rate was increased by about 10% by adding Hutat2:Fc containing medium from transduced hMDM (*P <0.05 vs. treatment with Tat alone). However, the rate was still lower than normal neurons, neurons treated with Tat86 plus HTB-Hutat2 medium, and Tat86 plus anti-Tat antibody (#P <0.01). Each value is the mean obtained from five random fields of three independent experiments using a 20× objective. Error bars denote the s.e.m. Scale bar = 100 μm.
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Fig4: Protection of the conditioned medium containing Hutat2:Fc against HIV-1 Tat86-mediated neurotoxicity in primary mouse neurons. Mouse cortical neurons cultured in 24-well plates were treated with HIV-1 Tat86 (Clade B, 500 nM) alone, or Tat with conditioned mediums from HR-Hutat2-transduced hMDM or HTB-11 (1:5 dilution) on day 6 in vitro (DIV 6) for 3 days. Treatment with Tat plus anti-Tat monoclonal antibody was used as a positive control, while Tat plus the conditioned medium from HR-A3H5 transduced HTB-11 was used as a negative control, respectively. (A) Representative images of primary mouse cortical neurons which were treated with HIV-1 Tat86 or Tat86 plus the conditioned medium from HR-Hutat2-transduced hMDM. Cells were counterstained with anti-MAP2 (MAP2), FITC-dUTP (TUNEL), and DAPI (Nuclei). Images of MAP2, TUNEL, and Nuclei were merged together (Merge). The survived neurons were the cells which were positive for MAP2 and DAPI but negative for TUNEL staining. Tat, Neurons treated with HIV-1 Tat86 alone; Tat/hMDM-Hutat2 medium, Neurons treated with HIV-1 Tat86 plus the conditioned medium of transduced hMDM; Normal control, Untreated neurons. Images were acquired as described in Figure 1. (B) Comparison of relative rates of neuron survival after treatment. The neuron survival rate of untreated neurons was defined as 100%. The relative neuron survival rate was increased by about 10% by adding Hutat2:Fc containing medium from transduced hMDM (*P <0.05 vs. treatment with Tat alone). However, the rate was still lower than normal neurons, neurons treated with Tat86 plus HTB-Hutat2 medium, and Tat86 plus anti-Tat antibody (#P <0.01). Each value is the mean obtained from five random fields of three independent experiments using a 20× objective. Error bars denote the s.e.m. Scale bar = 100 μm.
Mentions: In addition, these protective effects of Hutat2:Fc in the conditioned mediums were further evaluated using primary cultures of mouse neurons. Early postnatal (P0) Balb/c mouse neurons from cortex were isolated and cultured for 6 DIVs. The purity of the cultures were >95% neurons proved by MAP2 and glial fibrillary acidic protein immunocytochemistry staining (data not shown). The representative images of normal neurons and neurons treated with Tat86 or Tat86 plus Hutat2:Fc containing mediums from the transduced hMDM are shown in Figure 4A. Tat-treated mouse neurons showed increased numbers of cell apoptosis (Figure 4A TUNEL panel), loss of dendritic arbor, as well as a shorter dendrite length (Figure 4A; MAP2 panel). The relative rate of neuron survival was similar among normal neurons, neurons treated with Tat86 plus conditioned medium from HTB-Hutat2 (93.0 ± 4.5%), and neurons treated with Tat86 plus anti-Tat antibody (97.0 ± 7.2%). Compared with Tat exposure alone, the relative rate of neuron survival was increased by 10%, from 69.3 ± 8.9% to 79.4 ± 7.9% in the presence of conditioned medium from HR-Hutat2-transduced hMDM (P <0.05). However, the neuron survival rates were not significantly changed when adding HTB-A3H5 medium (66.6 ± 9.6% versus 69.3 ± 8.9%, P >0.05; Figure 4B). These results indicate that Hutat2:Fc released from transduced hMDM and HTB-11 could neutralize HIV-1 Tat86-induced neurotoxicity as an anti-Tat antibody in vitro, whereas A3H5:Fc released from HTB-A3H5 control does not have that biological effect. In comparison, the protective level of Hutat2:Fc from the conditioned medium of transduced hMDM was lower than that obtained from the use of transduced HTB-11 medium and the commercial anti-Tat antibody.Figure 4

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