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Functional proteomic analysis for regulatory T cell surveillance of the HIV-1-infected macrophage.

Huang X, Stone DK, Yu F, Zeng Y, Gendelman HE - J. Proteome Res. (2010)

Bottom Line: Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity.Taken together, Treg affects a range of virus-infected MP functions.The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States.

ABSTRACT
Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.

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Treg Uses Granzyme A, B and Perforin to Kill Virus-Infected BMM. (A) Western blotting demonstrating increased p38 phosphorylation (upper panel) and perforin insertion (middle panel), which were normalized to β-actin (bottom panel) in HIV-1/VSV infected BMM cocultured with Treg compared with HIV-1/VSV infected BMM cocultured with Tcon groups. Data shown are representative of three independent experiments. (B) Confocal immunofluorescences of BMM with specific monoclonal antibodies against granzyme A (upper panel), granzyme B (middle panel) and perforin (bottom panel) show cytoplasmic granular staining of granzyme A (red), granzyme B (red) and perforin (red) in Tcon and Treg treated groups. Both control and HIV-1/VSV infected groups showed no staining of those antibodies, which means BMM did not produce granzyme and perforin. Higher intensity of granzyme and perforin were seen in the Treg treated group compared with the Tcon treated group. Data shown are representative of three independent experiments. White scale bars represent 5 μm. (C) Cleaved NDUFS3, the 30 kDa subunit of mitochondrial complex I and the important substrate of granzyme A, was shown in HIV-1/VSV infected BMM cocultured with Treg group by Western blotting. Data shown are representative of three independent experiments. (D) Membrane potential-dependent staining of mitochondria in HIV-1/VSV infected BMM by JC-1 visualized by fluorescence microscopy showed the loss of red JC-1 aggregates fluorescence and cytoplasmic diffusion of green monomer fluorescence following coculture with Treg.
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fig4: Treg Uses Granzyme A, B and Perforin to Kill Virus-Infected BMM. (A) Western blotting demonstrating increased p38 phosphorylation (upper panel) and perforin insertion (middle panel), which were normalized to β-actin (bottom panel) in HIV-1/VSV infected BMM cocultured with Treg compared with HIV-1/VSV infected BMM cocultured with Tcon groups. Data shown are representative of three independent experiments. (B) Confocal immunofluorescences of BMM with specific monoclonal antibodies against granzyme A (upper panel), granzyme B (middle panel) and perforin (bottom panel) show cytoplasmic granular staining of granzyme A (red), granzyme B (red) and perforin (red) in Tcon and Treg treated groups. Both control and HIV-1/VSV infected groups showed no staining of those antibodies, which means BMM did not produce granzyme and perforin. Higher intensity of granzyme and perforin were seen in the Treg treated group compared with the Tcon treated group. Data shown are representative of three independent experiments. White scale bars represent 5 μm. (C) Cleaved NDUFS3, the 30 kDa subunit of mitochondrial complex I and the important substrate of granzyme A, was shown in HIV-1/VSV infected BMM cocultured with Treg group by Western blotting. Data shown are representative of three independent experiments. (D) Membrane potential-dependent staining of mitochondria in HIV-1/VSV infected BMM by JC-1 visualized by fluorescence microscopy showed the loss of red JC-1 aggregates fluorescence and cytoplasmic diffusion of green monomer fluorescence following coculture with Treg.

Mentions: To verify that Treg cells inserted perforin into the HIV-1-infected cell membrane, an important step in transferring granzymes to target cells, we used immunoblotting assays to detect inserted perforin. A very clear band was observed in Treg treated cell groups, while a diminished one was in Tcon treated macrophages (Figure 4A). Next, we used intracellular staining and confocal microscopy to visualize granzymes and perforin in BMM. Both control and HIV-1/VSV infected BMM showed no staining, indicating BMM do not produce granzyme and perforin de novo. However, cytoplasmic granular staining of granzymes and perforin were seen in the Treg treated cells and less so in Tcon BMM cocultivations (Figure 4B).


Functional proteomic analysis for regulatory T cell surveillance of the HIV-1-infected macrophage.

Huang X, Stone DK, Yu F, Zeng Y, Gendelman HE - J. Proteome Res. (2010)

Treg Uses Granzyme A, B and Perforin to Kill Virus-Infected BMM. (A) Western blotting demonstrating increased p38 phosphorylation (upper panel) and perforin insertion (middle panel), which were normalized to β-actin (bottom panel) in HIV-1/VSV infected BMM cocultured with Treg compared with HIV-1/VSV infected BMM cocultured with Tcon groups. Data shown are representative of three independent experiments. (B) Confocal immunofluorescences of BMM with specific monoclonal antibodies against granzyme A (upper panel), granzyme B (middle panel) and perforin (bottom panel) show cytoplasmic granular staining of granzyme A (red), granzyme B (red) and perforin (red) in Tcon and Treg treated groups. Both control and HIV-1/VSV infected groups showed no staining of those antibodies, which means BMM did not produce granzyme and perforin. Higher intensity of granzyme and perforin were seen in the Treg treated group compared with the Tcon treated group. Data shown are representative of three independent experiments. White scale bars represent 5 μm. (C) Cleaved NDUFS3, the 30 kDa subunit of mitochondrial complex I and the important substrate of granzyme A, was shown in HIV-1/VSV infected BMM cocultured with Treg group by Western blotting. Data shown are representative of three independent experiments. (D) Membrane potential-dependent staining of mitochondria in HIV-1/VSV infected BMM by JC-1 visualized by fluorescence microscopy showed the loss of red JC-1 aggregates fluorescence and cytoplasmic diffusion of green monomer fluorescence following coculture with Treg.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3108468&req=5

fig4: Treg Uses Granzyme A, B and Perforin to Kill Virus-Infected BMM. (A) Western blotting demonstrating increased p38 phosphorylation (upper panel) and perforin insertion (middle panel), which were normalized to β-actin (bottom panel) in HIV-1/VSV infected BMM cocultured with Treg compared with HIV-1/VSV infected BMM cocultured with Tcon groups. Data shown are representative of three independent experiments. (B) Confocal immunofluorescences of BMM with specific monoclonal antibodies against granzyme A (upper panel), granzyme B (middle panel) and perforin (bottom panel) show cytoplasmic granular staining of granzyme A (red), granzyme B (red) and perforin (red) in Tcon and Treg treated groups. Both control and HIV-1/VSV infected groups showed no staining of those antibodies, which means BMM did not produce granzyme and perforin. Higher intensity of granzyme and perforin were seen in the Treg treated group compared with the Tcon treated group. Data shown are representative of three independent experiments. White scale bars represent 5 μm. (C) Cleaved NDUFS3, the 30 kDa subunit of mitochondrial complex I and the important substrate of granzyme A, was shown in HIV-1/VSV infected BMM cocultured with Treg group by Western blotting. Data shown are representative of three independent experiments. (D) Membrane potential-dependent staining of mitochondria in HIV-1/VSV infected BMM by JC-1 visualized by fluorescence microscopy showed the loss of red JC-1 aggregates fluorescence and cytoplasmic diffusion of green monomer fluorescence following coculture with Treg.
Mentions: To verify that Treg cells inserted perforin into the HIV-1-infected cell membrane, an important step in transferring granzymes to target cells, we used immunoblotting assays to detect inserted perforin. A very clear band was observed in Treg treated cell groups, while a diminished one was in Tcon treated macrophages (Figure 4A). Next, we used intracellular staining and confocal microscopy to visualize granzymes and perforin in BMM. Both control and HIV-1/VSV infected BMM showed no staining, indicating BMM do not produce granzyme and perforin de novo. However, cytoplasmic granular staining of granzymes and perforin were seen in the Treg treated cells and less so in Tcon BMM cocultivations (Figure 4B).

Bottom Line: Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity.Taken together, Treg affects a range of virus-infected MP functions.The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States.

ABSTRACT
Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.

Show MeSH
Related in: MedlinePlus