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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 Transforms the Infected BMM. (A) Treg induces HIV-1/VSV infected BMM morphology change from elongation to roundness shown by confocal immunofluorescence microscopy (zoomed part is shown at the bottom panel). White scale bars represent 20 μm. (B) Western blotting showed Treg downregulated iNOS, while upregulated arginase-1. (C) The concentration of NO in supernatant was measured with Griess test. Treg inhibited NO production from HIV-1/VSV infected BMM, ***p < 0.001. (D) Concentrations of cAMP in the lysate (L) and supernatant (S) of Tcon or Treg were detected by ELISA, *p < 0.05 (E) Concentrations of cAMP in the lysate and supernatant of BMM with different treatments. The cAMP in the supernatant was undetectable, while its concentration in lysate increased in Treg treated group. ***p < 0.001.
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fig5: Treg Transforms the Infected BMM. (A) Treg induces HIV-1/VSV infected BMM morphology change from elongation to roundness shown by confocal immunofluorescence microscopy (zoomed part is shown at the bottom panel). White scale bars represent 20 μm. (B) Western blotting showed Treg downregulated iNOS, while upregulated arginase-1. (C) The concentration of NO in supernatant was measured with Griess test. Treg inhibited NO production from HIV-1/VSV infected BMM, ***p < 0.001. (D) Concentrations of cAMP in the lysate (L) and supernatant (S) of Tcon or Treg were detected by ELISA, *p < 0.05 (E) Concentrations of cAMP in the lysate and supernatant of BMM with different treatments. The cAMP in the supernatant was undetectable, while its concentration in lysate increased in Treg treated group. ***p < 0.001.

Mentions: We noted that among all cell death assays we used in this study, only around 50% of cells are killed by Treg. In addition, greater than 98% of BMM were infected as determined by p24 immunoflurescence staining (Supplemental Figure 1B, Supporting Information). So why did Treg only kill 50% of the cells? We observed that the privileged BMM underwent remarkable cytoskeletal rearrangement (Figure 5A), which may indicate its polarization switching.(40) As shown in Figure 5A, Treg induced changes in infected BMM morphology from a polarized to a rounded appearance with contracted processes showing redistribution of actin from focal adhesions to a cortical distribution. Such alterations paralleled functional outcomes in the BMM proteome (Table 1 and data not shown). We hypothesized that a polarization switch in this model could be due to either direct effects of Treg or in response to cytotoxicity induced by Treg. To test this, we used Western blotting to detect expression of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), which are putative markers for classical activation macrophage (M1) and alternative activation macrophage (M2), respectively. We found that Treg downregulated iNOS 35-fold, with a concomitant 18-fold upregulation of Arg-1 (Figure 5B). The decreased NO production by HIV-1/VSV-infected BMM cocultured with Treg supports this transformation. This is in stark contrast to the significantly increased NO production by infected BMM in the presence of Tcon (Figure 5C).


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 Transforms the Infected BMM. (A) Treg induces HIV-1/VSV infected BMM morphology change from elongation to roundness shown by confocal immunofluorescence microscopy (zoomed part is shown at the bottom panel). White scale bars represent 20 μm. (B) Western blotting showed Treg downregulated iNOS, while upregulated arginase-1. (C) The concentration of NO in supernatant was measured with Griess test. Treg inhibited NO production from HIV-1/VSV infected BMM, ***p < 0.001. (D) Concentrations of cAMP in the lysate (L) and supernatant (S) of Tcon or Treg were detected by ELISA, *p < 0.05 (E) Concentrations of cAMP in the lysate and supernatant of BMM with different treatments. The cAMP in the supernatant was undetectable, while its concentration in lysate increased in Treg treated group. ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Treg Transforms the Infected BMM. (A) Treg induces HIV-1/VSV infected BMM morphology change from elongation to roundness shown by confocal immunofluorescence microscopy (zoomed part is shown at the bottom panel). White scale bars represent 20 μm. (B) Western blotting showed Treg downregulated iNOS, while upregulated arginase-1. (C) The concentration of NO in supernatant was measured with Griess test. Treg inhibited NO production from HIV-1/VSV infected BMM, ***p < 0.001. (D) Concentrations of cAMP in the lysate (L) and supernatant (S) of Tcon or Treg were detected by ELISA, *p < 0.05 (E) Concentrations of cAMP in the lysate and supernatant of BMM with different treatments. The cAMP in the supernatant was undetectable, while its concentration in lysate increased in Treg treated group. ***p < 0.001.
Mentions: We noted that among all cell death assays we used in this study, only around 50% of cells are killed by Treg. In addition, greater than 98% of BMM were infected as determined by p24 immunoflurescence staining (Supplemental Figure 1B, Supporting Information). So why did Treg only kill 50% of the cells? We observed that the privileged BMM underwent remarkable cytoskeletal rearrangement (Figure 5A), which may indicate its polarization switching.(40) As shown in Figure 5A, Treg induced changes in infected BMM morphology from a polarized to a rounded appearance with contracted processes showing redistribution of actin from focal adhesions to a cortical distribution. Such alterations paralleled functional outcomes in the BMM proteome (Table 1 and data not shown). We hypothesized that a polarization switch in this model could be due to either direct effects of Treg or in response to cytotoxicity induced by Treg. To test this, we used Western blotting to detect expression of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), which are putative markers for classical activation macrophage (M1) and alternative activation macrophage (M2), respectively. We found that Treg downregulated iNOS 35-fold, with a concomitant 18-fold upregulation of Arg-1 (Figure 5B). The decreased NO production by HIV-1/VSV-infected BMM cocultured with Treg supports this transformation. This is in stark contrast to the significantly increased NO production by infected BMM in the presence of Tcon (Figure 5C).

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