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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 and Tcon Induced Virus-Infected BMM Apoptosis and Pyroptosis. (A) Western blotting showed HIV-1/VSV infection triggers caspase-1 activation in BMM, which was enhanced by Tcon coculture while attenuated by Treg coculture. Data shown are representative of five independent experiments. (B) Western blotting showed Treg coculture activated caspase-3 pathway in HIV-1/VSV infection BMM. Data shown are representative of five independent experiments. (C) Caspase-1 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. Caspase-3 and 7 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. (D) Proposed mechanisms Treg and Tcon use to modulate HIV-1-infected macrophage functions. Upon infection, HIV-1 activates NF-κB and the inflammasome, which results in IL-1β release. Tcon worsened this response by secretion of proinflammatory cytokines. In addition, those proinflammatory cytokines increase mitochondrial activity, which could lead to energy exhaustion and pyroptosis. However, Treg use their anti-inflammatory cytokines to extinguish the inflammation resulting from virus infection. Treg induce infected BMM apoptosis. Compared with apoptosis, pyroptosis causes more ATP release. Excessive ATP is an important inflammatory molecule and could be hydrolyzed by ecto-nucleoside triphosphate diphosphohydrolase CD39 and CD73 expressed on Treg. Moreover, Treg could transport cAMP to infected BMM and contribute to inflammation resolution. The activation status of infected macrophage affects Th0 cell differentiation. For example, M1 induce Th1 and Th17, while M2 induce Th2 and iTreg. (E) Comparison of CD39 expression on Tcon and Treg was determined by flow cytometric analysis.
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fig3: Treg and Tcon Induced Virus-Infected BMM Apoptosis and Pyroptosis. (A) Western blotting showed HIV-1/VSV infection triggers caspase-1 activation in BMM, which was enhanced by Tcon coculture while attenuated by Treg coculture. Data shown are representative of five independent experiments. (B) Western blotting showed Treg coculture activated caspase-3 pathway in HIV-1/VSV infection BMM. Data shown are representative of five independent experiments. (C) Caspase-1 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. Caspase-3 and 7 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. (D) Proposed mechanisms Treg and Tcon use to modulate HIV-1-infected macrophage functions. Upon infection, HIV-1 activates NF-κB and the inflammasome, which results in IL-1β release. Tcon worsened this response by secretion of proinflammatory cytokines. In addition, those proinflammatory cytokines increase mitochondrial activity, which could lead to energy exhaustion and pyroptosis. However, Treg use their anti-inflammatory cytokines to extinguish the inflammation resulting from virus infection. Treg induce infected BMM apoptosis. Compared with apoptosis, pyroptosis causes more ATP release. Excessive ATP is an important inflammatory molecule and could be hydrolyzed by ecto-nucleoside triphosphate diphosphohydrolase CD39 and CD73 expressed on Treg. Moreover, Treg could transport cAMP to infected BMM and contribute to inflammation resolution. The activation status of infected macrophage affects Th0 cell differentiation. For example, M1 induce Th1 and Th17, while M2 induce Th2 and iTreg. (E) Comparison of CD39 expression on Tcon and Treg was determined by flow cytometric analysis.

Mentions: Based on these observations of Treg-induced control of viral infection, we next investigated molecular mechanisms underlying the Treg induced cytotoxic responses. Immunoblots for cleavage of procaspases-1 and 3 demonstrated that Treg induced HIV-1/VSV infected BMM apoptosis is caspase-3 specific. In contrast, Tcon induces infected BMM pyroptosis through caspase-1 (Figure 3A, B). HIV-1/VSV infection itself triggered caspase-1 activation in BMM and was enhanced by Tcon but was reduced by Treg (Figure 3A). Densitometric analysis of blots for caspase-1 subunits failed to reveal differences between caspase-1 p20 in HIV-1/VSV infected BMM alone versus infected BMM cocultured with Tcon (16 to 18% when compared to actin). However, there were differences in caspase-1 p10 observed between HIV-1/VSV infected BMM alone and HIV-1/VSV infected BMM cocultured with Tcon (11 to 17% when compared to actin). Futhermore, procaspase-1 was present in cell lysates from HIV-1/VSV infected BMM at 30% compared to actin, while 42% compared to actin from HIV-1/VSV infected BMM cocultured with Tcon. (Figure 3A). In addition, faint bands were observed in these blots for caspase-1 in the Treg treatment group and for caspase-3 in the Tcon treatement group, suggesting much limited activation. Apoptosis induction by Treg and pyroptosis by Tcon was detected independently using a Fluorochrome Inhibitor of Caspases Assay (FLICA) utilizing binding of fluorescent inhibitors to specific active caspases (Figure 3C). Replicate observations were also seen in human cells (Supplemental Figure 4A, Supporting Information).


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 and Tcon Induced Virus-Infected BMM Apoptosis and Pyroptosis. (A) Western blotting showed HIV-1/VSV infection triggers caspase-1 activation in BMM, which was enhanced by Tcon coculture while attenuated by Treg coculture. Data shown are representative of five independent experiments. (B) Western blotting showed Treg coculture activated caspase-3 pathway in HIV-1/VSV infection BMM. Data shown are representative of five independent experiments. (C) Caspase-1 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. Caspase-3 and 7 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. (D) Proposed mechanisms Treg and Tcon use to modulate HIV-1-infected macrophage functions. Upon infection, HIV-1 activates NF-κB and the inflammasome, which results in IL-1β release. Tcon worsened this response by secretion of proinflammatory cytokines. In addition, those proinflammatory cytokines increase mitochondrial activity, which could lead to energy exhaustion and pyroptosis. However, Treg use their anti-inflammatory cytokines to extinguish the inflammation resulting from virus infection. Treg induce infected BMM apoptosis. Compared with apoptosis, pyroptosis causes more ATP release. Excessive ATP is an important inflammatory molecule and could be hydrolyzed by ecto-nucleoside triphosphate diphosphohydrolase CD39 and CD73 expressed on Treg. Moreover, Treg could transport cAMP to infected BMM and contribute to inflammation resolution. The activation status of infected macrophage affects Th0 cell differentiation. For example, M1 induce Th1 and Th17, while M2 induce Th2 and iTreg. (E) Comparison of CD39 expression on Tcon and Treg was determined by flow cytometric analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Treg and Tcon Induced Virus-Infected BMM Apoptosis and Pyroptosis. (A) Western blotting showed HIV-1/VSV infection triggers caspase-1 activation in BMM, which was enhanced by Tcon coculture while attenuated by Treg coculture. Data shown are representative of five independent experiments. (B) Western blotting showed Treg coculture activated caspase-3 pathway in HIV-1/VSV infection BMM. Data shown are representative of five independent experiments. (C) Caspase-1 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. Caspase-3 and 7 activation was measured by FLICA assay plus immunofluorescence microscopy. White scale bars represent 50 μm. (D) Proposed mechanisms Treg and Tcon use to modulate HIV-1-infected macrophage functions. Upon infection, HIV-1 activates NF-κB and the inflammasome, which results in IL-1β release. Tcon worsened this response by secretion of proinflammatory cytokines. In addition, those proinflammatory cytokines increase mitochondrial activity, which could lead to energy exhaustion and pyroptosis. However, Treg use their anti-inflammatory cytokines to extinguish the inflammation resulting from virus infection. Treg induce infected BMM apoptosis. Compared with apoptosis, pyroptosis causes more ATP release. Excessive ATP is an important inflammatory molecule and could be hydrolyzed by ecto-nucleoside triphosphate diphosphohydrolase CD39 and CD73 expressed on Treg. Moreover, Treg could transport cAMP to infected BMM and contribute to inflammation resolution. The activation status of infected macrophage affects Th0 cell differentiation. For example, M1 induce Th1 and Th17, while M2 induce Th2 and iTreg. (E) Comparison of CD39 expression on Tcon and Treg was determined by flow cytometric analysis.
Mentions: Based on these observations of Treg-induced control of viral infection, we next investigated molecular mechanisms underlying the Treg induced cytotoxic responses. Immunoblots for cleavage of procaspases-1 and 3 demonstrated that Treg induced HIV-1/VSV infected BMM apoptosis is caspase-3 specific. In contrast, Tcon induces infected BMM pyroptosis through caspase-1 (Figure 3A, B). HIV-1/VSV infection itself triggered caspase-1 activation in BMM and was enhanced by Tcon but was reduced by Treg (Figure 3A). Densitometric analysis of blots for caspase-1 subunits failed to reveal differences between caspase-1 p20 in HIV-1/VSV infected BMM alone versus infected BMM cocultured with Tcon (16 to 18% when compared to actin). However, there were differences in caspase-1 p10 observed between HIV-1/VSV infected BMM alone and HIV-1/VSV infected BMM cocultured with Tcon (11 to 17% when compared to actin). Futhermore, procaspase-1 was present in cell lysates from HIV-1/VSV infected BMM at 30% compared to actin, while 42% compared to actin from HIV-1/VSV infected BMM cocultured with Tcon. (Figure 3A). In addition, faint bands were observed in these blots for caspase-1 in the Treg treatment group and for caspase-3 in the Tcon treatement group, suggesting much limited activation. Apoptosis induction by Treg and pyroptosis by Tcon was detected independently using a Fluorochrome Inhibitor of Caspases Assay (FLICA) utilizing binding of fluorescent inhibitors to specific active caspases (Figure 3C). Replicate observations were also seen in human cells (Supplemental Figure 4A, Supporting Information).

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