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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 Akt.A, MDM incubated with or without HIV-1 Tat (1 ng/ml) were examined for expression of total Akt and phospho-Akt by immunoblot. B, MonoMac6 cells were transfected with siRNA against Akt1 or control siRNA. Expression of Akt and actin were examined 72 h post-transfection. C, MonoMac6 cells transfected with control or siRNA directed against Akt1 were transfected with LC3-eGFP. Cells were incubated with or without HIV-1 Tat (100 pg/ml) overnight followed by stimulation with rapamycin. Autophagy was assessed by examining expression of large LC3+ structures. D, MonoMac6 cells were transfected with control siRNA or siRNA directed against Akt. Cells were transfected with LC3-eGFP and incubated with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3+ structures. E, MDM were treated with Akt inhibitor IV (1.25 µM) or vehicle followed by incubation with HIV-1 Tat. Macrophages were incubated with or without CD154, challenged with T. gondii and assessed for parasite load at 24 h. Data are representative of 3 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.
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pone-0011733-g003: HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through Akt.A, MDM incubated with or without HIV-1 Tat (1 ng/ml) were examined for expression of total Akt and phospho-Akt by immunoblot. B, MonoMac6 cells were transfected with siRNA against Akt1 or control siRNA. Expression of Akt and actin were examined 72 h post-transfection. C, MonoMac6 cells transfected with control or siRNA directed against Akt1 were transfected with LC3-eGFP. Cells were incubated with or without HIV-1 Tat (100 pg/ml) overnight followed by stimulation with rapamycin. Autophagy was assessed by examining expression of large LC3+ structures. D, MonoMac6 cells were transfected with control siRNA or siRNA directed against Akt. Cells were transfected with LC3-eGFP and incubated with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3+ structures. E, MDM were treated with Akt inhibitor IV (1.25 µM) or vehicle followed by incubation with HIV-1 Tat. Macrophages were incubated with or without CD154, challenged with T. gondii and assessed for parasite load at 24 h. Data are representative of 3 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.

Mentions: HIV-1 can activate Akt [32], [33], which is an inhibitor of autophagy [34], [35]. To determine if Akt mediates the inhibitory effects of Tat on autophagy, we initially examined if Tat induced activation of Akt in macrophages. MDM exhibited enhanced phosphorylation of Akt when incubated with HIV-1 Tat (Figure 3A). Next, we determined if Akt signaling was necessary for the inhibition of autophagy induced by HIV-1 Tat in bystander cells. To this end, we silenced Akt in MonoMac6 cells by utilizing siRNA (Figure 3B). Fluorescent microscopy of LC3-eGFP+ MonoMac6 cells revealed that HIV-1 Tat was unable to inhibit autophagy when these cells were transfected with Akt1 siRNA (Figure 3C). Additionally, cells infected with pseudotyped HIV-1 were no longer able to inhibit autophagy in MonoMac6 cells that had silenced Akt1 (Figure 3D). Similar results were obtained with Akt inhibitor IV (Figure S2A). Incubation with Akt inhibitor IV also ablated the ability of HIV-1 Tat to block CD40-dependent autophagic killing of T. gondii (Figure 3E). These results indicate that HIV-1 inhibits autophagy in bystander primary macrophage and monocytic cells through an Akt-dependent pathway.


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 Akt.A, MDM incubated with or without HIV-1 Tat (1 ng/ml) were examined for expression of total Akt and phospho-Akt by immunoblot. B, MonoMac6 cells were transfected with siRNA against Akt1 or control siRNA. Expression of Akt and actin were examined 72 h post-transfection. C, MonoMac6 cells transfected with control or siRNA directed against Akt1 were transfected with LC3-eGFP. Cells were incubated with or without HIV-1 Tat (100 pg/ml) overnight followed by stimulation with rapamycin. Autophagy was assessed by examining expression of large LC3+ structures. D, MonoMac6 cells were transfected with control siRNA or siRNA directed against Akt. Cells were transfected with LC3-eGFP and incubated with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3+ structures. E, MDM were treated with Akt inhibitor IV (1.25 µM) or vehicle followed by incubation with HIV-1 Tat. Macrophages were incubated with or without CD154, challenged with T. gondii and assessed for parasite load at 24 h. Data are representative of 3 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.
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pone-0011733-g003: HIV-1 inhibits autophagy in bystander macrophages/monocytic cells through Akt.A, MDM incubated with or without HIV-1 Tat (1 ng/ml) were examined for expression of total Akt and phospho-Akt by immunoblot. B, MonoMac6 cells were transfected with siRNA against Akt1 or control siRNA. Expression of Akt and actin were examined 72 h post-transfection. C, MonoMac6 cells transfected with control or siRNA directed against Akt1 were transfected with LC3-eGFP. Cells were incubated with or without HIV-1 Tat (100 pg/ml) overnight followed by stimulation with rapamycin. Autophagy was assessed by examining expression of large LC3+ structures. D, MonoMac6 cells were transfected with control siRNA or siRNA directed against Akt. Cells were transfected with LC3-eGFP and incubated with MonoMac6 cells infected with pseudotyped control virus (PV Ctr) or pseudotyped HIV-1 (PV HIV). Cultures were treated with or without rapamycin. Autophagy was assessed by examining expression of large LC3+ structures. E, MDM were treated with Akt inhibitor IV (1.25 µM) or vehicle followed by incubation with HIV-1 Tat. Macrophages were incubated with or without CD154, challenged with T. gondii and assessed for parasite load at 24 h. Data are representative of 3 independent experiments presented as means ± SEM; *p≤0.05, **p≤0.01, ∧p≥0.05.
Mentions: HIV-1 can activate Akt [32], [33], which is an inhibitor of autophagy [34], [35]. To determine if Akt mediates the inhibitory effects of Tat on autophagy, we initially examined if Tat induced activation of Akt in macrophages. MDM exhibited enhanced phosphorylation of Akt when incubated with HIV-1 Tat (Figure 3A). Next, we determined if Akt signaling was necessary for the inhibition of autophagy induced by HIV-1 Tat in bystander cells. To this end, we silenced Akt in MonoMac6 cells by utilizing siRNA (Figure 3B). Fluorescent microscopy of LC3-eGFP+ MonoMac6 cells revealed that HIV-1 Tat was unable to inhibit autophagy when these cells were transfected with Akt1 siRNA (Figure 3C). Additionally, cells infected with pseudotyped HIV-1 were no longer able to inhibit autophagy in MonoMac6 cells that had silenced Akt1 (Figure 3D). Similar results were obtained with Akt inhibitor IV (Figure S2A). Incubation with Akt inhibitor IV also ablated the ability of HIV-1 Tat to block CD40-dependent autophagic killing of T. gondii (Figure 3E). These results indicate that HIV-1 inhibits autophagy in bystander primary macrophage and monocytic cells through an Akt-dependent pathway.

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