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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.A and B, MDM from healthy controls and HIV-1+ patients were incubated with or without CD154 (3 µg/ml) (A) or IFN-γ (200 U/ml) (B) followed by challenge with T. gondii tachyzoites. The number of parasites per 100 macrophages was assessed by light microscopy 24 h post-challenge. HIV-1+ patients were classified as non-responder when their macrophages exhibited a percentage decrease in parasite load that was less than the 10th percentile of the percentage decrease in parasite load observed in macrophages from healthy controls. C–E, MDM from healthy controls were infected with pseudotyped HIV-1 (PV HIV) or pseudotyped control virus (PV Ctr) and were incubated with a monolayer of uninfected macrophages. In certain experiments macrophages were treated with or without zidovudine (AZT) 2 h after incubation with pseudotyped HIV-1 (E). Macrophage monolayers were treated with or without CD154 followed by challenge with T. gondii and assessment of parasite load at 24 h. F, Schematic representation of MonoMac6 cells infected with pseudotyped HIV-1 (PV HIV) or pseudotyped control virus (PV Ctr) incubated with uninfected MonoMac6 cells transfected with LC3-eGFP. Cells were treated with or without rapamycin (1 µM) and assessed for autophagy by expression of large (≥1 µm) LC3+ structures. G, Flourescent images of LC3-eGFP+ cells as treated in panel (F). H, Quantification of autophagic cells as treated in panel (F). Data are representative of 4 independent experiments presented as means ± SEM; ***p≤0.001; **p≤0.01; *≤0.05; ∧p≥0.05.
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pone-0011733-g001: HIV-1 inhibits autophagy in bystander macrophages/monocytic cells.A and B, MDM from healthy controls and HIV-1+ patients were incubated with or without CD154 (3 µg/ml) (A) or IFN-γ (200 U/ml) (B) followed by challenge with T. gondii tachyzoites. The number of parasites per 100 macrophages was assessed by light microscopy 24 h post-challenge. HIV-1+ patients were classified as non-responder when their macrophages exhibited a percentage decrease in parasite load that was less than the 10th percentile of the percentage decrease in parasite load observed in macrophages from healthy controls. C–E, MDM from healthy controls were infected with pseudotyped HIV-1 (PV HIV) or pseudotyped control virus (PV Ctr) and were incubated with a monolayer of uninfected macrophages. In certain experiments macrophages were treated with or without zidovudine (AZT) 2 h after incubation with pseudotyped HIV-1 (E). Macrophage monolayers were treated with or without CD154 followed by challenge with T. gondii and assessment of parasite load at 24 h. F, Schematic representation of MonoMac6 cells infected with pseudotyped HIV-1 (PV HIV) or pseudotyped control virus (PV Ctr) incubated with uninfected MonoMac6 cells transfected with LC3-eGFP. Cells were treated with or without rapamycin (1 µM) and assessed for autophagy by expression of large (≥1 µm) LC3+ structures. G, Flourescent images of LC3-eGFP+ cells as treated in panel (F). H, Quantification of autophagic cells as treated in panel (F). Data are representative of 4 independent experiments presented as means ± SEM; ***p≤0.001; **p≤0.01; *≤0.05; ∧p≥0.05.

Mentions: CD40 stimulation of macrophages results in killing of T. gondii through autophagy [11], [12]. To determine whether autophagy is affected by HIV-1, we examined autophagic killing of T. gondii in macrophages from HIV-1+ patients. Monocyte-derived macrophages (MDM) from HIV-1+ patients and healthy controls were incubated with or without CD154 (CD40 ligand) followed by challenge with T. gondii. MDM from 5 of a total of 23 HIV-1+ patients exhibited defective induction of anti-T. gondii activity (Figure 1A). Impaired anti-T. gondii activity was reproducible since similar results were obtained when MDM were retested 2 to 3 different times. The group of HIV-1+ patients with defective CD40-induced toxoplasmacidal response will be referred to as CD40 non-responders. CD40-induced anti-T. gondii activity did not correlate with CD4 counts or plasma viral load (R = 0.02; p = 0.76; data not shown). Impaired CD40-dependent toxoplasmacidal activity was not due to differences in CD40 expression on MDM (CD40 corrected mean fluorescence intensity: controls 26.6±4.4; CD40 responders 29.9±4.5; CD40 non-responders 32.6±6.4; p>0.1). While CD40 induces T. gondii killing through autophagy, IFN-γ works independently of autophagy to kill T. gondii [11]. Indeed, in marked contrast to results obtained with CD40-stimulated MDM, anti-T. gondii activity induced by IFN-γ was similar in all groups (Figure 1B).


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.A and B, MDM from healthy controls and HIV-1+ patients were incubated with or without CD154 (3 µg/ml) (A) or IFN-γ (200 U/ml) (B) followed by challenge with T. gondii tachyzoites. The number of parasites per 100 macrophages was assessed by light microscopy 24 h post-challenge. HIV-1+ patients were classified as non-responder when their macrophages exhibited a percentage decrease in parasite load that was less than the 10th percentile of the percentage decrease in parasite load observed in macrophages from healthy controls. C–E, MDM from healthy controls were infected with pseudotyped HIV-1 (PV HIV) or pseudotyped control virus (PV Ctr) and were incubated with a monolayer of uninfected macrophages. In certain experiments macrophages were treated with or without zidovudine (AZT) 2 h after incubation with pseudotyped HIV-1 (E). Macrophage monolayers were treated with or without CD154 followed by challenge with T. gondii and assessment of parasite load at 24 h. F, Schematic representation of MonoMac6 cells infected with pseudotyped HIV-1 (PV HIV) or pseudotyped control virus (PV Ctr) incubated with uninfected MonoMac6 cells transfected with LC3-eGFP. Cells were treated with or without rapamycin (1 µM) and assessed for autophagy by expression of large (≥1 µm) LC3+ structures. G, Flourescent images of LC3-eGFP+ cells as treated in panel (F). H, Quantification of autophagic cells as treated in panel (F). Data are representative of 4 independent experiments presented as means ± SEM; ***p≤0.001; **p≤0.01; *≤0.05; ∧p≥0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908694&req=5

pone-0011733-g001: HIV-1 inhibits autophagy in bystander macrophages/monocytic cells.A and B, MDM from healthy controls and HIV-1+ patients were incubated with or without CD154 (3 µg/ml) (A) or IFN-γ (200 U/ml) (B) followed by challenge with T. gondii tachyzoites. The number of parasites per 100 macrophages was assessed by light microscopy 24 h post-challenge. HIV-1+ patients were classified as non-responder when their macrophages exhibited a percentage decrease in parasite load that was less than the 10th percentile of the percentage decrease in parasite load observed in macrophages from healthy controls. C–E, MDM from healthy controls were infected with pseudotyped HIV-1 (PV HIV) or pseudotyped control virus (PV Ctr) and were incubated with a monolayer of uninfected macrophages. In certain experiments macrophages were treated with or without zidovudine (AZT) 2 h after incubation with pseudotyped HIV-1 (E). Macrophage monolayers were treated with or without CD154 followed by challenge with T. gondii and assessment of parasite load at 24 h. F, Schematic representation of MonoMac6 cells infected with pseudotyped HIV-1 (PV HIV) or pseudotyped control virus (PV Ctr) incubated with uninfected MonoMac6 cells transfected with LC3-eGFP. Cells were treated with or without rapamycin (1 µM) and assessed for autophagy by expression of large (≥1 µm) LC3+ structures. G, Flourescent images of LC3-eGFP+ cells as treated in panel (F). H, Quantification of autophagic cells as treated in panel (F). Data are representative of 4 independent experiments presented as means ± SEM; ***p≤0.001; **p≤0.01; *≤0.05; ∧p≥0.05.
Mentions: CD40 stimulation of macrophages results in killing of T. gondii through autophagy [11], [12]. To determine whether autophagy is affected by HIV-1, we examined autophagic killing of T. gondii in macrophages from HIV-1+ patients. Monocyte-derived macrophages (MDM) from HIV-1+ patients and healthy controls were incubated with or without CD154 (CD40 ligand) followed by challenge with T. gondii. MDM from 5 of a total of 23 HIV-1+ patients exhibited defective induction of anti-T. gondii activity (Figure 1A). Impaired anti-T. gondii activity was reproducible since similar results were obtained when MDM were retested 2 to 3 different times. The group of HIV-1+ patients with defective CD40-induced toxoplasmacidal response will be referred to as CD40 non-responders. CD40-induced anti-T. gondii activity did not correlate with CD4 counts or plasma viral load (R = 0.02; p = 0.76; data not shown). Impaired CD40-dependent toxoplasmacidal activity was not due to differences in CD40 expression on MDM (CD40 corrected mean fluorescence intensity: controls 26.6±4.4; CD40 responders 29.9±4.5; CD40 non-responders 32.6±6.4; p>0.1). While CD40 induces T. gondii killing through autophagy, IFN-γ works independently of autophagy to kill T. gondii [11]. Indeed, in marked contrast to results obtained with CD40-stimulated MDM, anti-T. gondii activity induced by IFN-γ was similar in all groups (Figure 1B).

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