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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 inhibition of virus release from infected BMM is interferon-stimulated gene 15 pathway associated. (A) Western blotting showed increased STAT1 phosphorylation in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (B) Western blotting showed dramatically increased free ISG15 and ISGylated proteins in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (C) Western blotting showed no change of HIV-1 p24 expression between HIV-1/VSV infected BMM group, HIV-1/VSV infected BMM group cocultured with Tcon group and HIV-1/VSV infected BMM group cocultured with Treg group. In this study the pseudotyped virus we used could enter the mouse BMM once, so the level of p24 staining is dependent only on the infective dose. Data shown are representative of three independent experiments. (D) Both Tcon and Treg could inhibit HIV-1 release determined by HIV-1 p24 ELISA. Data shown are representative of three independent experiments. ***p < 0.001. (E) Proposed pathways involved in initiating antiviral immune response in HIV-1-infected macrophages, which was enhanced by Treg and Tcon. In macrophages, engagement of TLR3 by HIV derived dsRNA in endosomes mediates IFN-α/β/λ production. TLR3 induces a Trif-dependent pathway, which recruits kinases (TBK1, IKKϵ and IKKαβγ) that mediate activation of the transcription factors IRF3 and NF-κB. Together with IRF7, IRF3 and NF-κB translocate into the nucleus and bind to positive regulatory domains on the Ifn genes promoter, leading to Ifn transcription. Secreted IFNs signal through binding to their cognate receptors (IFNAR1 and IFNAR2) and then recruit kinases (TYK2 and JAK1) leading to translocation of phosphorylated transcription factors (STAT1 and STAT2) and IRF9 to the nucleus where binding to the enhancer region of the ifn promoter and isg promoter occurs, which leads to the activation and nuclear transport of ISGF3 and induction of ISRE to produce IFNs and ISGs. Both IFNs and ISGs could inhibit ubiquitination of HIV Gag and Tsg101, which results in less virus release. With unknown mechanisms, Treg exhibit much greater capacity to enhance the antiviral immune response than Tcon.
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fig1: Treg inhibition of virus release from infected BMM is interferon-stimulated gene 15 pathway associated. (A) Western blotting showed increased STAT1 phosphorylation in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (B) Western blotting showed dramatically increased free ISG15 and ISGylated proteins in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (C) Western blotting showed no change of HIV-1 p24 expression between HIV-1/VSV infected BMM group, HIV-1/VSV infected BMM group cocultured with Tcon group and HIV-1/VSV infected BMM group cocultured with Treg group. In this study the pseudotyped virus we used could enter the mouse BMM once, so the level of p24 staining is dependent only on the infective dose. Data shown are representative of three independent experiments. (D) Both Tcon and Treg could inhibit HIV-1 release determined by HIV-1 p24 ELISA. Data shown are representative of three independent experiments. ***p < 0.001. (E) Proposed pathways involved in initiating antiviral immune response in HIV-1-infected macrophages, which was enhanced by Treg and Tcon. In macrophages, engagement of TLR3 by HIV derived dsRNA in endosomes mediates IFN-α/β/λ production. TLR3 induces a Trif-dependent pathway, which recruits kinases (TBK1, IKKϵ and IKKαβγ) that mediate activation of the transcription factors IRF3 and NF-κB. Together with IRF7, IRF3 and NF-κB translocate into the nucleus and bind to positive regulatory domains on the Ifn genes promoter, leading to Ifn transcription. Secreted IFNs signal through binding to their cognate receptors (IFNAR1 and IFNAR2) and then recruit kinases (TYK2 and JAK1) leading to translocation of phosphorylated transcription factors (STAT1 and STAT2) and IRF9 to the nucleus where binding to the enhancer region of the ifn promoter and isg promoter occurs, which leads to the activation and nuclear transport of ISGF3 and induction of ISRE to produce IFNs and ISGs. Both IFNs and ISGs could inhibit ubiquitination of HIV Gag and Tsg101, which results in less virus release. With unknown mechanisms, Treg exhibit much greater capacity to enhance the antiviral immune response than Tcon.

Mentions: To elucidate the mechanisms by which Treg could alter functional outcomes for HIV-1-infected macrophages, we first focused on validating the enhanced antiviral immune response supported by protein changes observed after strict filtering of the iTRAQ data. Treg coculture resulted in upregulation of BMM expression of signal transducer and activator of transcription 1 (STAT1) and ISG15 (Figure 1A, B) found in type I IFN antiviral signaling.(34) Since ISG15 was reported to inhibit release of HIV-1 virions, we assayed whether the increased ISG15 expression observed resulted in reduced virion release.(34) HIV-1p24 levels in BMM cell lysates and culture supernatants were measured by Western blotting and ELISA. No changes in HIV-1p24 were observed between HIV-1/VSV infected BMMs and HIV-1/VSV infected BMMs cocultured with CD3/CD28-activated Treg or Tcon in cell lysates (Figure 1C). This reflected that the pseudotyped virus is not capable of spreading viral infection, so the level of HIV-1p24 is dependent only on the initial infective dose (Supplemental Figures 1B, C, Supporting Information). However, ELISA tests of culture supernatant fluids showed both Tcon and Treg inhibited virus release by reductions in HIV-1p24 concentrations (Figure 1D). Moreover, Treg more than Tcon (the inhibition rate is 79 versus 45%, respectively) inhibited HIV-1p24 release in culture fluids. We posit that such findings could result from stimulation of autocrine/paracrine pathways linked to innate IFN antiviral immunity regulated by Treg (Figure 1E).


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 inhibition of virus release from infected BMM is interferon-stimulated gene 15 pathway associated. (A) Western blotting showed increased STAT1 phosphorylation in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (B) Western blotting showed dramatically increased free ISG15 and ISGylated proteins in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (C) Western blotting showed no change of HIV-1 p24 expression between HIV-1/VSV infected BMM group, HIV-1/VSV infected BMM group cocultured with Tcon group and HIV-1/VSV infected BMM group cocultured with Treg group. In this study the pseudotyped virus we used could enter the mouse BMM once, so the level of p24 staining is dependent only on the infective dose. Data shown are representative of three independent experiments. (D) Both Tcon and Treg could inhibit HIV-1 release determined by HIV-1 p24 ELISA. Data shown are representative of three independent experiments. ***p < 0.001. (E) Proposed pathways involved in initiating antiviral immune response in HIV-1-infected macrophages, which was enhanced by Treg and Tcon. In macrophages, engagement of TLR3 by HIV derived dsRNA in endosomes mediates IFN-α/β/λ production. TLR3 induces a Trif-dependent pathway, which recruits kinases (TBK1, IKKϵ and IKKαβγ) that mediate activation of the transcription factors IRF3 and NF-κB. Together with IRF7, IRF3 and NF-κB translocate into the nucleus and bind to positive regulatory domains on the Ifn genes promoter, leading to Ifn transcription. Secreted IFNs signal through binding to their cognate receptors (IFNAR1 and IFNAR2) and then recruit kinases (TYK2 and JAK1) leading to translocation of phosphorylated transcription factors (STAT1 and STAT2) and IRF9 to the nucleus where binding to the enhancer region of the ifn promoter and isg promoter occurs, which leads to the activation and nuclear transport of ISGF3 and induction of ISRE to produce IFNs and ISGs. Both IFNs and ISGs could inhibit ubiquitination of HIV Gag and Tsg101, which results in less virus release. With unknown mechanisms, Treg exhibit much greater capacity to enhance the antiviral immune response than Tcon.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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fig1: Treg inhibition of virus release from infected BMM is interferon-stimulated gene 15 pathway associated. (A) Western blotting showed increased STAT1 phosphorylation in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (B) Western blotting showed dramatically increased free ISG15 and ISGylated proteins in HIV-1/VSV infected BMM cocultured with Treg group. Data shown are representative of three independent experiments. (C) Western blotting showed no change of HIV-1 p24 expression between HIV-1/VSV infected BMM group, HIV-1/VSV infected BMM group cocultured with Tcon group and HIV-1/VSV infected BMM group cocultured with Treg group. In this study the pseudotyped virus we used could enter the mouse BMM once, so the level of p24 staining is dependent only on the infective dose. Data shown are representative of three independent experiments. (D) Both Tcon and Treg could inhibit HIV-1 release determined by HIV-1 p24 ELISA. Data shown are representative of three independent experiments. ***p < 0.001. (E) Proposed pathways involved in initiating antiviral immune response in HIV-1-infected macrophages, which was enhanced by Treg and Tcon. In macrophages, engagement of TLR3 by HIV derived dsRNA in endosomes mediates IFN-α/β/λ production. TLR3 induces a Trif-dependent pathway, which recruits kinases (TBK1, IKKϵ and IKKαβγ) that mediate activation of the transcription factors IRF3 and NF-κB. Together with IRF7, IRF3 and NF-κB translocate into the nucleus and bind to positive regulatory domains on the Ifn genes promoter, leading to Ifn transcription. Secreted IFNs signal through binding to their cognate receptors (IFNAR1 and IFNAR2) and then recruit kinases (TYK2 and JAK1) leading to translocation of phosphorylated transcription factors (STAT1 and STAT2) and IRF9 to the nucleus where binding to the enhancer region of the ifn promoter and isg promoter occurs, which leads to the activation and nuclear transport of ISGF3 and induction of ISRE to produce IFNs and ISGs. Both IFNs and ISGs could inhibit ubiquitination of HIV Gag and Tsg101, which results in less virus release. With unknown mechanisms, Treg exhibit much greater capacity to enhance the antiviral immune response than Tcon.
Mentions: To elucidate the mechanisms by which Treg could alter functional outcomes for HIV-1-infected macrophages, we first focused on validating the enhanced antiviral immune response supported by protein changes observed after strict filtering of the iTRAQ data. Treg coculture resulted in upregulation of BMM expression of signal transducer and activator of transcription 1 (STAT1) and ISG15 (Figure 1A, B) found in type I IFN antiviral signaling.(34) Since ISG15 was reported to inhibit release of HIV-1 virions, we assayed whether the increased ISG15 expression observed resulted in reduced virion release.(34) HIV-1p24 levels in BMM cell lysates and culture supernatants were measured by Western blotting and ELISA. No changes in HIV-1p24 were observed between HIV-1/VSV infected BMMs and HIV-1/VSV infected BMMs cocultured with CD3/CD28-activated Treg or Tcon in cell lysates (Figure 1C). This reflected that the pseudotyped virus is not capable of spreading viral infection, so the level of HIV-1p24 is dependent only on the initial infective dose (Supplemental Figures 1B, C, Supporting Information). However, ELISA tests of culture supernatant fluids showed both Tcon and Treg inhibited virus release by reductions in HIV-1p24 concentrations (Figure 1D). Moreover, Treg more than Tcon (the inhibition rate is 79 versus 45%, respectively) inhibited HIV-1p24 release in culture fluids. We posit that such findings could result from stimulation of autocrine/paracrine pathways linked to innate IFN antiviral immunity regulated by Treg (Figure 1E).

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