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HIV-1 inhibits autophagy in bystander macrophage/monocytic cells through Src-Akt and STAT3.

Van Grol J, Subauste C, Andrade RM, Fujinaga K, Nelson J, Subauste CS - PLoS ONE (2010)

Bottom Line: Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy.These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways.The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America.

ABSTRACT
Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, beta-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1(+) patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.

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HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through CXCR4, VEGFR and β-integrin.A, MonoMa6 cells were transfected with LC3-eGFP and incubated with cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated overnight with control mAb, control peptide cRAD, anti-CXCR4 mAb, anti-VEGFR1 mAb, VEGFR1 tyrosine kinase inhibitor, or β-integrin blocking peptide cRGD. MonoMac6 cells were treated with or with out rapamycin and assessed for large LC3+ structures. B, MonoMac6 cells transfected with LC3-eGFP were treated overnight with or without Tat (100 pg/ml) and either control mAb, control peptide cRAD, anti-CXCR4 mAb, anti-VEGFR1 mAb or β-integrin blocking peptide, cRGD. MonoMac6 cells were treated with or without rapamycin and assessed for large LC3+ structures. C, Schematic representation of MonoMac6 cells transfected with LC3-eGFP cultured overnight with MonoMac6 cells transfected with a control plasmid or a plasmid encoding Tat. Cultures were treated with rapamycin and assessed for large LC3+ structures. D, MonoMac6 cells transfected with LC3-eGFP were cultured overnight with MonoMac6 cells transfected with a control plasmid or a plasmid encoding either wild-type Tat, Tat CD26 mutant, Tat chemokine-like domain mutant (Tat31–101), Tat basic domain mutant, or Tat RGD mutant as depicted in panel (C). Cultures were treated with rapamycin and assessed for large LC3+ structures. Data are representative of 4 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.
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pone-0011733-g005: HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through CXCR4, VEGFR and β-integrin.A, MonoMa6 cells were transfected with LC3-eGFP and incubated with cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated overnight with control mAb, control peptide cRAD, anti-CXCR4 mAb, anti-VEGFR1 mAb, VEGFR1 tyrosine kinase inhibitor, or β-integrin blocking peptide cRGD. MonoMac6 cells were treated with or with out rapamycin and assessed for large LC3+ structures. B, MonoMac6 cells transfected with LC3-eGFP were treated overnight with or without Tat (100 pg/ml) and either control mAb, control peptide cRAD, anti-CXCR4 mAb, anti-VEGFR1 mAb or β-integrin blocking peptide, cRGD. MonoMac6 cells were treated with or without rapamycin and assessed for large LC3+ structures. C, Schematic representation of MonoMac6 cells transfected with LC3-eGFP cultured overnight with MonoMac6 cells transfected with a control plasmid or a plasmid encoding Tat. Cultures were treated with rapamycin and assessed for large LC3+ structures. D, MonoMac6 cells transfected with LC3-eGFP were cultured overnight with MonoMac6 cells transfected with a control plasmid or a plasmid encoding either wild-type Tat, Tat CD26 mutant, Tat chemokine-like domain mutant (Tat31–101), Tat basic domain mutant, or Tat RGD mutant as depicted in panel (C). Cultures were treated with rapamycin and assessed for large LC3+ structures. Data are representative of 4 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.

Mentions: Since HIV-1 Tat increased phosphorylation of Src and FAK in macrophages, we examined the role of receptors known to signal through these molecules: chemokine receptors, VEGFR and β-integrin [37], [38], [39]. To this end, we blocked engagement of these surface molecules on LC3-eGFP+ MonoMac6 cells prior to incubation with pseudotyped HIV-1 infected cells and treatment with rapamycin. A blocking mAb against CXCR4 was used because CXCR4 is a predominant chemokine receptor on macrophages [40] and treatment of MonoMac6 cells with recombinant SDF, the ligand for CXCR4, caused inhibition of autophagy (Figure S3). VEGFR signaling was inhibited with a mAb against VEGFR1 (the predominant form of VEGFR on macrophages), or a VEGFR1 tyrosine-kinase inhibitor. β-integrin signaling was inhibited with a blocking RGD peptide. Neutralization of any of these molecules impaired the ability of cells infected with pseudotyped HIV-1 to inhibit autophagy in bystander cells (Figure 5A). Similar results were obtained with HIV-1 Tat (Figure 5B). These results suggest that all three cell surface molecules are required for successful inhibition of autophagy by HIV-1.


HIV-1 inhibits autophagy in bystander macrophage/monocytic cells through Src-Akt and STAT3.

Van Grol J, Subauste C, Andrade RM, Fujinaga K, Nelson J, Subauste CS - PLoS ONE (2010)

HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through CXCR4, VEGFR and β-integrin.A, MonoMa6 cells were transfected with LC3-eGFP and incubated with cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated overnight with control mAb, control peptide cRAD, anti-CXCR4 mAb, anti-VEGFR1 mAb, VEGFR1 tyrosine kinase inhibitor, or β-integrin blocking peptide cRGD. MonoMac6 cells were treated with or with out rapamycin and assessed for large LC3+ structures. B, MonoMac6 cells transfected with LC3-eGFP were treated overnight with or without Tat (100 pg/ml) and either control mAb, control peptide cRAD, anti-CXCR4 mAb, anti-VEGFR1 mAb or β-integrin blocking peptide, cRGD. MonoMac6 cells were treated with or without rapamycin and assessed for large LC3+ structures. C, Schematic representation of MonoMac6 cells transfected with LC3-eGFP cultured overnight with MonoMac6 cells transfected with a control plasmid or a plasmid encoding Tat. Cultures were treated with rapamycin and assessed for large LC3+ structures. D, MonoMac6 cells transfected with LC3-eGFP were cultured overnight with MonoMac6 cells transfected with a control plasmid or a plasmid encoding either wild-type Tat, Tat CD26 mutant, Tat chemokine-like domain mutant (Tat31–101), Tat basic domain mutant, or Tat RGD mutant as depicted in panel (C). Cultures were treated with rapamycin and assessed for large LC3+ structures. Data are representative of 4 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.
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getmorefigures.php?uid=PMC2908694&req=5

pone-0011733-g005: HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through CXCR4, VEGFR and β-integrin.A, MonoMa6 cells were transfected with LC3-eGFP and incubated with cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated overnight with control mAb, control peptide cRAD, anti-CXCR4 mAb, anti-VEGFR1 mAb, VEGFR1 tyrosine kinase inhibitor, or β-integrin blocking peptide cRGD. MonoMac6 cells were treated with or with out rapamycin and assessed for large LC3+ structures. B, MonoMac6 cells transfected with LC3-eGFP were treated overnight with or without Tat (100 pg/ml) and either control mAb, control peptide cRAD, anti-CXCR4 mAb, anti-VEGFR1 mAb or β-integrin blocking peptide, cRGD. MonoMac6 cells were treated with or without rapamycin and assessed for large LC3+ structures. C, Schematic representation of MonoMac6 cells transfected with LC3-eGFP cultured overnight with MonoMac6 cells transfected with a control plasmid or a plasmid encoding Tat. Cultures were treated with rapamycin and assessed for large LC3+ structures. D, MonoMac6 cells transfected with LC3-eGFP were cultured overnight with MonoMac6 cells transfected with a control plasmid or a plasmid encoding either wild-type Tat, Tat CD26 mutant, Tat chemokine-like domain mutant (Tat31–101), Tat basic domain mutant, or Tat RGD mutant as depicted in panel (C). Cultures were treated with rapamycin and assessed for large LC3+ structures. Data are representative of 4 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.
Mentions: Since HIV-1 Tat increased phosphorylation of Src and FAK in macrophages, we examined the role of receptors known to signal through these molecules: chemokine receptors, VEGFR and β-integrin [37], [38], [39]. To this end, we blocked engagement of these surface molecules on LC3-eGFP+ MonoMac6 cells prior to incubation with pseudotyped HIV-1 infected cells and treatment with rapamycin. A blocking mAb against CXCR4 was used because CXCR4 is a predominant chemokine receptor on macrophages [40] and treatment of MonoMac6 cells with recombinant SDF, the ligand for CXCR4, caused inhibition of autophagy (Figure S3). VEGFR signaling was inhibited with a mAb against VEGFR1 (the predominant form of VEGFR on macrophages), or a VEGFR1 tyrosine-kinase inhibitor. β-integrin signaling was inhibited with a blocking RGD peptide. Neutralization of any of these molecules impaired the ability of cells infected with pseudotyped HIV-1 to inhibit autophagy in bystander cells (Figure 5A). Similar results were obtained with HIV-1 Tat (Figure 5B). These results suggest that all three cell surface molecules are required for successful inhibition of autophagy by HIV-1.

Bottom Line: Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy.These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways.The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America.

ABSTRACT
Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, beta-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1(+) patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.

Show MeSH
Related in: MedlinePlus