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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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The effects of transduction with lentiviral vector HR-Hutat2 on the gene expression of human macrophage-related functional and regulatory genes and on kinetics of pro-inflammatory cytokines IL1β, IL8, IL10, and TNF-α. Human monocyte-derived macrophages (hMDM) were differentiated from isolated peripheral blood mononuclear cells in M-CSF-containing medium. On day 7 and day 8 in vitro (DIV 7 and DIV 8), hMDMs were transduced with HR-Hutat2 vector at a MOI of 10 or 50. Total RNA was extracted from non-transduced hMDM (Normal) and transduced hMDM on day 9 post-transduction. Cell culture mediums were collected every 3 days post-transduction. (A) Comparative analysis of the transcriptional profiling of 15 hMDM-related functional and regulatory genes by qRT-PCR. Among the 15 genes, only the transcription of IL8, STAT1, and IDO1 genes changed. (B–E) Sequential changes of IL1β, IL10, IL8, and TNF-α levels in the supernatants of normal and transduced hMDMs at a MOI of 10 or 50. Normal, Non-transduced hMDM; MOI 10, hMDM transduced with HR-Hutat2 at the MOI of 10; MOI 50, hMDM transduced with HR-Hutat2 at the MOI of 50. *P <0.01, #P <0.05 compared with normal. Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.
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Fig6: The effects of transduction with lentiviral vector HR-Hutat2 on the gene expression of human macrophage-related functional and regulatory genes and on kinetics of pro-inflammatory cytokines IL1β, IL8, IL10, and TNF-α. Human monocyte-derived macrophages (hMDM) were differentiated from isolated peripheral blood mononuclear cells in M-CSF-containing medium. On day 7 and day 8 in vitro (DIV 7 and DIV 8), hMDMs were transduced with HR-Hutat2 vector at a MOI of 10 or 50. Total RNA was extracted from non-transduced hMDM (Normal) and transduced hMDM on day 9 post-transduction. Cell culture mediums were collected every 3 days post-transduction. (A) Comparative analysis of the transcriptional profiling of 15 hMDM-related functional and regulatory genes by qRT-PCR. Among the 15 genes, only the transcription of IL8, STAT1, and IDO1 genes changed. (B–E) Sequential changes of IL1β, IL10, IL8, and TNF-α levels in the supernatants of normal and transduced hMDMs at a MOI of 10 or 50. Normal, Non-transduced hMDM; MOI 10, hMDM transduced with HR-Hutat2 at the MOI of 10; MOI 50, hMDM transduced with HR-Hutat2 at the MOI of 50. *P <0.01, #P <0.05 compared with normal. Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.

Mentions: Second, a qRT-PCR assay was employed to comparatively evaluate the expression of 15 human macrophage-related pro-inflammatory cytokine genes, apoptosis-related genes, tumor-related genes, cell signal transduction genes, and cell surface receptor genes (Table 1) between normal and HR-Hutat2-transduced hMDM on day 9 post-transduction. Differential gene expression was considered “significant” when the normalized fold change of samples versus control was >2 (up-regulated) or <0.5 (down-regulated). Twelve out of 15 genes retained their expression at the same level in transduced hMDM at a MOI of 10 or 50 compared with normal hMDM (Figure 6A). However, the change of gene expression level was detected in three genes, IL8, STAT1, and indoleamine-pyrrole 2,3-dioxygenase (IDO)1. STAT1 was 3.36 ± 0.34-fold up-regulated in the MOI 10 group and 4.29 ± 0.77-fold up-regulated in the MOI 50 group as compared to non-transduced hMDM (P <0.01). It was 326.8 ± 56.5- and 409.3 ± 86.3-fold up-regulated for IDO1 gene expression level in transduced hMDM at a MOI of 10 and 50, respectively (P <0.01). The expression of IL8 increased by 5.2 ± 1.2-fold for the transduction at a MOI of 50 (P <0.01) as compared to non-transduced hMDM.Figure 6


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)

The effects of transduction with lentiviral vector HR-Hutat2 on the gene expression of human macrophage-related functional and regulatory genes and on kinetics of pro-inflammatory cytokines IL1β, IL8, IL10, and TNF-α. Human monocyte-derived macrophages (hMDM) were differentiated from isolated peripheral blood mononuclear cells in M-CSF-containing medium. On day 7 and day 8 in vitro (DIV 7 and DIV 8), hMDMs were transduced with HR-Hutat2 vector at a MOI of 10 or 50. Total RNA was extracted from non-transduced hMDM (Normal) and transduced hMDM on day 9 post-transduction. Cell culture mediums were collected every 3 days post-transduction. (A) Comparative analysis of the transcriptional profiling of 15 hMDM-related functional and regulatory genes by qRT-PCR. Among the 15 genes, only the transcription of IL8, STAT1, and IDO1 genes changed. (B–E) Sequential changes of IL1β, IL10, IL8, and TNF-α levels in the supernatants of normal and transduced hMDMs at a MOI of 10 or 50. Normal, Non-transduced hMDM; MOI 10, hMDM transduced with HR-Hutat2 at the MOI of 10; MOI 50, hMDM transduced with HR-Hutat2 at the MOI of 50. *P <0.01, #P <0.05 compared with normal. Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: The effects of transduction with lentiviral vector HR-Hutat2 on the gene expression of human macrophage-related functional and regulatory genes and on kinetics of pro-inflammatory cytokines IL1β, IL8, IL10, and TNF-α. Human monocyte-derived macrophages (hMDM) were differentiated from isolated peripheral blood mononuclear cells in M-CSF-containing medium. On day 7 and day 8 in vitro (DIV 7 and DIV 8), hMDMs were transduced with HR-Hutat2 vector at a MOI of 10 or 50. Total RNA was extracted from non-transduced hMDM (Normal) and transduced hMDM on day 9 post-transduction. Cell culture mediums were collected every 3 days post-transduction. (A) Comparative analysis of the transcriptional profiling of 15 hMDM-related functional and regulatory genes by qRT-PCR. Among the 15 genes, only the transcription of IL8, STAT1, and IDO1 genes changed. (B–E) Sequential changes of IL1β, IL10, IL8, and TNF-α levels in the supernatants of normal and transduced hMDMs at a MOI of 10 or 50. Normal, Non-transduced hMDM; MOI 10, hMDM transduced with HR-Hutat2 at the MOI of 10; MOI 50, hMDM transduced with HR-Hutat2 at the MOI of 50. *P <0.01, #P <0.05 compared with normal. Results shown represent mean values from three independent experiments. Error bars denote the s.e.m.
Mentions: Second, a qRT-PCR assay was employed to comparatively evaluate the expression of 15 human macrophage-related pro-inflammatory cytokine genes, apoptosis-related genes, tumor-related genes, cell signal transduction genes, and cell surface receptor genes (Table 1) between normal and HR-Hutat2-transduced hMDM on day 9 post-transduction. Differential gene expression was considered “significant” when the normalized fold change of samples versus control was >2 (up-regulated) or <0.5 (down-regulated). Twelve out of 15 genes retained their expression at the same level in transduced hMDM at a MOI of 10 or 50 compared with normal hMDM (Figure 6A). However, the change of gene expression level was detected in three genes, IL8, STAT1, and indoleamine-pyrrole 2,3-dioxygenase (IDO)1. STAT1 was 3.36 ± 0.34-fold up-regulated in the MOI 10 group and 4.29 ± 0.77-fold up-regulated in the MOI 50 group as compared to non-transduced hMDM (P <0.01). It was 326.8 ± 56.5- and 409.3 ± 86.3-fold up-regulated for IDO1 gene expression level in transduced hMDM at a MOI of 10 and 50, respectively (P <0.01). The expression of IL8 increased by 5.2 ± 1.2-fold for the transduction at a MOI of 50 (P <0.01) as compared to non-transduced hMDM.Figure 6

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