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Suppression of Foxo1 activity and down-modulation of CD62L (L-selectin) in HIV-1 infected resting CD4 T cells.

Trinité B, Chan CN, Lee CS, Mahajan S, Luo Y, Muesing MA, Folkvord JM, Pham M, Connick E, Levy DN - PLoS ONE (2014)

Bottom Line: CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation.Partial T cell activation was further evident as an increase in CD69 expression.Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Science, New York University College of Dentistry, New York, New York, United States of America.

ABSTRACT
HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection in vitro, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized in vitro to study HIV-1 replication in resting CD4 T cells. In vivo, the migration of resting naïve and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naïve and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated de novo viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication in vivo.

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CD62L down-modulation is not the result of apoptosis, virus contact or protease cleavage, but is influenced by PI3K.A. Cells infected with for 5 days, then were analyzed for GFP, CD62L and Annexin V surface expression, and permeability to 7-AAD. Data are representative of >5 independent experiments. B. Contact with HIV-1 virions does not affect CD62L surface expression. Cells were treated with the reverse transcriptase inhibitor efavirenz (EFV) (2 µg/ml) and infected by spinoculation or spinoculated without virus (Mock). Surface CD62L expression was analyzed at the indicated times after infection. A positive control infection was performed without EFV as indicated. EFV completely prevented the appearance of GFP+ cells (not shown). C. The PI3K and mTORC2 inhibitors LY294002 and PI-103 suppress HIV-1-induced CD62L down-modulation. Infected cells were treated with LY294002 (5 µM), PI-103 (5 µM) or a carrier control (DMSO) 1 day and again 4 hours prior to staining for CD62L. Middle graph: Data from 3 independent experiments with different cell donors. Y axis = the mean fluorescence intensity of CD62L staining (MFI) for each condition expressed as a percentage of the CD62L MFI of the GFP-negative cells. Right: GFP expression is modestly suppressed by LY294002 and PI-103 (MFI = 1761 and 1471 respectively, vs.1981 for DMSO control). LY294002 and PI-103 treatments were statistically different (p<0.01) from their respective DMSO controls by a T(x) population comparison test [134]. D. Metalloprotease inhibitor (MTPI) TAPI-1 (50 µM) does not inhibit HIV-1-induced CD62L surface down-modulation. Left: Control experiment demonstrating inhibition of PMA-induced CD62L downmodulation by MTPI. Cells were treated with PMA with and without MTPI and analyzed one hour later. Right: Cells infected with HIV-1 GFP reporter virus were treated with MTPI 1 day and again 3 hours prior to staining. Data are representative of 2 independent experiments.
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pone-0110719-g002: CD62L down-modulation is not the result of apoptosis, virus contact or protease cleavage, but is influenced by PI3K.A. Cells infected with for 5 days, then were analyzed for GFP, CD62L and Annexin V surface expression, and permeability to 7-AAD. Data are representative of >5 independent experiments. B. Contact with HIV-1 virions does not affect CD62L surface expression. Cells were treated with the reverse transcriptase inhibitor efavirenz (EFV) (2 µg/ml) and infected by spinoculation or spinoculated without virus (Mock). Surface CD62L expression was analyzed at the indicated times after infection. A positive control infection was performed without EFV as indicated. EFV completely prevented the appearance of GFP+ cells (not shown). C. The PI3K and mTORC2 inhibitors LY294002 and PI-103 suppress HIV-1-induced CD62L down-modulation. Infected cells were treated with LY294002 (5 µM), PI-103 (5 µM) or a carrier control (DMSO) 1 day and again 4 hours prior to staining for CD62L. Middle graph: Data from 3 independent experiments with different cell donors. Y axis = the mean fluorescence intensity of CD62L staining (MFI) for each condition expressed as a percentage of the CD62L MFI of the GFP-negative cells. Right: GFP expression is modestly suppressed by LY294002 and PI-103 (MFI = 1761 and 1471 respectively, vs.1981 for DMSO control). LY294002 and PI-103 treatments were statistically different (p<0.01) from their respective DMSO controls by a T(x) population comparison test [134]. D. Metalloprotease inhibitor (MTPI) TAPI-1 (50 µM) does not inhibit HIV-1-induced CD62L surface down-modulation. Left: Control experiment demonstrating inhibition of PMA-induced CD62L downmodulation by MTPI. Cells were treated with PMA with and without MTPI and analyzed one hour later. Right: Cells infected with HIV-1 GFP reporter virus were treated with MTPI 1 day and again 3 hours prior to staining. Data are representative of 2 independent experiments.

Mentions: To explore the mechanism(s) responsible for HIV-1-induced CD62L down-modulation, we first tested whether apoptosis of GFP+ cells was inducing CD62L shedding [70], [71]. However, Annexin V and 7-AAD staining were very low (0.9%) on GFP+ cells that were down-modulating or had down-modulated CD62L (Figure 2A). Prior studies have reported that HIV-1 binding to cells can induce ADAM17-dependent shedding of CD62L through the interaction between envelope protein and CD4 or CXCR4 [72], [73], while another study reported upregulation [74]. To test whether virus binding influenced CD62L expression in our system, we stained cells soon after spinoculation of virus onto cells. Infection was performed in the presence of the reverse transcriptase inhibitor efavirenz (EFV) in order to block events downstream of virus binding and entry. No effect on CD62L expression was observed at any time from 4 hours to 5 days after infection in the presence of EFV (Figure 2B). It has also been reported that contact between Jurkat T cells infected with an Envelope wild type virus and uninfected primary cells led to CD62L shedding [72], but in a separate test we observed no CD62L loss by this method either (data not shown). Failure of coculture of infected and uninfected cells to affect CD62L expression is consistent with the results in Figure 1A that CD62L down-modulation was restricted to the productively infected GFP+ cells and was not observed on GFP-negative bystander cells.


Suppression of Foxo1 activity and down-modulation of CD62L (L-selectin) in HIV-1 infected resting CD4 T cells.

Trinité B, Chan CN, Lee CS, Mahajan S, Luo Y, Muesing MA, Folkvord JM, Pham M, Connick E, Levy DN - PLoS ONE (2014)

CD62L down-modulation is not the result of apoptosis, virus contact or protease cleavage, but is influenced by PI3K.A. Cells infected with for 5 days, then were analyzed for GFP, CD62L and Annexin V surface expression, and permeability to 7-AAD. Data are representative of >5 independent experiments. B. Contact with HIV-1 virions does not affect CD62L surface expression. Cells were treated with the reverse transcriptase inhibitor efavirenz (EFV) (2 µg/ml) and infected by spinoculation or spinoculated without virus (Mock). Surface CD62L expression was analyzed at the indicated times after infection. A positive control infection was performed without EFV as indicated. EFV completely prevented the appearance of GFP+ cells (not shown). C. The PI3K and mTORC2 inhibitors LY294002 and PI-103 suppress HIV-1-induced CD62L down-modulation. Infected cells were treated with LY294002 (5 µM), PI-103 (5 µM) or a carrier control (DMSO) 1 day and again 4 hours prior to staining for CD62L. Middle graph: Data from 3 independent experiments with different cell donors. Y axis = the mean fluorescence intensity of CD62L staining (MFI) for each condition expressed as a percentage of the CD62L MFI of the GFP-negative cells. Right: GFP expression is modestly suppressed by LY294002 and PI-103 (MFI = 1761 and 1471 respectively, vs.1981 for DMSO control). LY294002 and PI-103 treatments were statistically different (p<0.01) from their respective DMSO controls by a T(x) population comparison test [134]. D. Metalloprotease inhibitor (MTPI) TAPI-1 (50 µM) does not inhibit HIV-1-induced CD62L surface down-modulation. Left: Control experiment demonstrating inhibition of PMA-induced CD62L downmodulation by MTPI. Cells were treated with PMA with and without MTPI and analyzed one hour later. Right: Cells infected with HIV-1 GFP reporter virus were treated with MTPI 1 day and again 3 hours prior to staining. Data are representative of 2 independent experiments.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4199762&req=5

pone-0110719-g002: CD62L down-modulation is not the result of apoptosis, virus contact or protease cleavage, but is influenced by PI3K.A. Cells infected with for 5 days, then were analyzed for GFP, CD62L and Annexin V surface expression, and permeability to 7-AAD. Data are representative of >5 independent experiments. B. Contact with HIV-1 virions does not affect CD62L surface expression. Cells were treated with the reverse transcriptase inhibitor efavirenz (EFV) (2 µg/ml) and infected by spinoculation or spinoculated without virus (Mock). Surface CD62L expression was analyzed at the indicated times after infection. A positive control infection was performed without EFV as indicated. EFV completely prevented the appearance of GFP+ cells (not shown). C. The PI3K and mTORC2 inhibitors LY294002 and PI-103 suppress HIV-1-induced CD62L down-modulation. Infected cells were treated with LY294002 (5 µM), PI-103 (5 µM) or a carrier control (DMSO) 1 day and again 4 hours prior to staining for CD62L. Middle graph: Data from 3 independent experiments with different cell donors. Y axis = the mean fluorescence intensity of CD62L staining (MFI) for each condition expressed as a percentage of the CD62L MFI of the GFP-negative cells. Right: GFP expression is modestly suppressed by LY294002 and PI-103 (MFI = 1761 and 1471 respectively, vs.1981 for DMSO control). LY294002 and PI-103 treatments were statistically different (p<0.01) from their respective DMSO controls by a T(x) population comparison test [134]. D. Metalloprotease inhibitor (MTPI) TAPI-1 (50 µM) does not inhibit HIV-1-induced CD62L surface down-modulation. Left: Control experiment demonstrating inhibition of PMA-induced CD62L downmodulation by MTPI. Cells were treated with PMA with and without MTPI and analyzed one hour later. Right: Cells infected with HIV-1 GFP reporter virus were treated with MTPI 1 day and again 3 hours prior to staining. Data are representative of 2 independent experiments.
Mentions: To explore the mechanism(s) responsible for HIV-1-induced CD62L down-modulation, we first tested whether apoptosis of GFP+ cells was inducing CD62L shedding [70], [71]. However, Annexin V and 7-AAD staining were very low (0.9%) on GFP+ cells that were down-modulating or had down-modulated CD62L (Figure 2A). Prior studies have reported that HIV-1 binding to cells can induce ADAM17-dependent shedding of CD62L through the interaction between envelope protein and CD4 or CXCR4 [72], [73], while another study reported upregulation [74]. To test whether virus binding influenced CD62L expression in our system, we stained cells soon after spinoculation of virus onto cells. Infection was performed in the presence of the reverse transcriptase inhibitor efavirenz (EFV) in order to block events downstream of virus binding and entry. No effect on CD62L expression was observed at any time from 4 hours to 5 days after infection in the presence of EFV (Figure 2B). It has also been reported that contact between Jurkat T cells infected with an Envelope wild type virus and uninfected primary cells led to CD62L shedding [72], but in a separate test we observed no CD62L loss by this method either (data not shown). Failure of coculture of infected and uninfected cells to affect CD62L expression is consistent with the results in Figure 1A that CD62L down-modulation was restricted to the productively infected GFP+ cells and was not observed on GFP-negative bystander cells.

Bottom Line: CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation.Partial T cell activation was further evident as an increase in CD69 expression.Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Science, New York University College of Dentistry, New York, New York, United States of America.

ABSTRACT
HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection in vitro, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized in vitro to study HIV-1 replication in resting CD4 T cells. In vivo, the migration of resting naïve and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naïve and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated de novo viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication in vivo.

Show MeSH
Related in: MedlinePlus