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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 Induces Virus-Infected BMM Death. (A) Treg induces HIV-1/VSV-infected BMM death shown by TUNEL assay plus confocal immunofluorescence microscopy and trypan blue staining plus light microscopy. For TUNEL assay, bright field is shown in the top panel and the corresponding fluorescent field is shown in the middle panel. Trypan blue staining is shown in the bottom panel (trypan blue positive cells are indicated by yellow arrows). White scale bar represent 100 μm. (B) Graphs represent the mean percentage of TUNEL positive BMM and trypan blue positive BMM counted and calculated from five different fields (data were pooled from three independent experiments). Each bar represents a mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001.
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fig2: Treg Induces Virus-Infected BMM Death. (A) Treg induces HIV-1/VSV-infected BMM death shown by TUNEL assay plus confocal immunofluorescence microscopy and trypan blue staining plus light microscopy. For TUNEL assay, bright field is shown in the top panel and the corresponding fluorescent field is shown in the middle panel. Trypan blue staining is shown in the bottom panel (trypan blue positive cells are indicated by yellow arrows). White scale bar represent 100 μm. (B) Graphs represent the mean percentage of TUNEL positive BMM and trypan blue positive BMM counted and calculated from five different fields (data were pooled from three independent experiments). Each bar represents a mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: In the past decade following Treg discovery, substantive research has focused on the mechanisms for immune tolerance by Treg through its inhibitory molecule expression and secretion, such as CTLA-4, GITR, IL-10 and TGF-β (reviewed by Zhu and Paul(35)). However, Tregs also exhibit perforin-dependent cytotoxicity against autologous target cells(36) indicating that Treg can act as natural cytotoxic cells, like natural killer cells and cytotoxic T lymphocytes. Based on such prior findings and our observations of granzymes in the infected BMM lysates treated with Treg by mass spectrometry, we hypothesized that Treg may induce HIV-1-infected macrophage death to deplete the cell source of neurotoxic factors. To test this hypothesis, we used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to assess the cytotoxicity of Treg against HIV-1/VSV infected BMM. Greater than 50% of BMMs were TUNEL positive in Treg treated cocultures (Figure 2A, B). The findings mirrored trypan blue exclusion tests (Figure 2A, B). Cytotoxicity of Treg was <10% in uninfected BMM (data not shown) supporting the notion that Treg are killers of activated or pathogen-altered BMM. Results were replicated in HIV-1ADA infected human MDM cocultured with human Treg (Supplemental Figure 4A−C, Supporting Information). In these assays human MDM were shrunken and detached following Treg cocultivation (Supplemental Figure 4C, Supporting Information). Taken together, the reductions seen in HIV-1p24 in macrophage culture supernatant fluids by Treg likely reflect both antiviral innate immunity and the killing of infected cells.


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 Induces Virus-Infected BMM Death. (A) Treg induces HIV-1/VSV-infected BMM death shown by TUNEL assay plus confocal immunofluorescence microscopy and trypan blue staining plus light microscopy. For TUNEL assay, bright field is shown in the top panel and the corresponding fluorescent field is shown in the middle panel. Trypan blue staining is shown in the bottom panel (trypan blue positive cells are indicated by yellow arrows). White scale bar represent 100 μm. (B) Graphs represent the mean percentage of TUNEL positive BMM and trypan blue positive BMM counted and calculated from five different fields (data were pooled from three independent experiments). Each bar represents a mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Treg Induces Virus-Infected BMM Death. (A) Treg induces HIV-1/VSV-infected BMM death shown by TUNEL assay plus confocal immunofluorescence microscopy and trypan blue staining plus light microscopy. For TUNEL assay, bright field is shown in the top panel and the corresponding fluorescent field is shown in the middle panel. Trypan blue staining is shown in the bottom panel (trypan blue positive cells are indicated by yellow arrows). White scale bar represent 100 μm. (B) Graphs represent the mean percentage of TUNEL positive BMM and trypan blue positive BMM counted and calculated from five different fields (data were pooled from three independent experiments). Each bar represents a mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: In the past decade following Treg discovery, substantive research has focused on the mechanisms for immune tolerance by Treg through its inhibitory molecule expression and secretion, such as CTLA-4, GITR, IL-10 and TGF-β (reviewed by Zhu and Paul(35)). However, Tregs also exhibit perforin-dependent cytotoxicity against autologous target cells(36) indicating that Treg can act as natural cytotoxic cells, like natural killer cells and cytotoxic T lymphocytes. Based on such prior findings and our observations of granzymes in the infected BMM lysates treated with Treg by mass spectrometry, we hypothesized that Treg may induce HIV-1-infected macrophage death to deplete the cell source of neurotoxic factors. To test this hypothesis, we used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to assess the cytotoxicity of Treg against HIV-1/VSV infected BMM. Greater than 50% of BMMs were TUNEL positive in Treg treated cocultures (Figure 2A, B). The findings mirrored trypan blue exclusion tests (Figure 2A, B). Cytotoxicity of Treg was <10% in uninfected BMM (data not shown) supporting the notion that Treg are killers of activated or pathogen-altered BMM. Results were replicated in HIV-1ADA infected human MDM cocultured with human Treg (Supplemental Figure 4A−C, Supporting Information). In these assays human MDM were shrunken and detached following Treg cocultivation (Supplemental Figure 4C, Supporting Information). Taken together, the reductions seen in HIV-1p24 in macrophage culture supernatant fluids by Treg likely reflect both antiviral innate immunity and the killing of infected cells.

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