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Mammalian Target of Rapamycin Inhibition in Macrophages of Asymptomatic HIV+ Persons Reverses the Decrease in TLR-4–Mediated TNF-α Release through Prolongation of MAPK Pathway Activation

Xin Li, Xinbing Han, Juliana Llano, Medhavi Bole, Xiuqin Zhou, Katharine Swan, Asha Anandaiah, Benjamin Nelson, Naimish R. Patel, Peter S. Reinach, Henry Koziel and Souvenir D. Tachado
J Immunol December 1, 2011, 187 (11) 6052-6058; DOI: https://doi.org/10.4049/jimmunol.1101532
Xin Li
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Xinbing Han
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Juliana Llano
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Medhavi Bole
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Xiuqin Zhou
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Katharine Swan
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Asha Anandaiah
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Benjamin Nelson
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Naimish R. Patel
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Peter S. Reinach
†Department of Biological Sciences, State University of New York, College of Optometry, New York, NY 10036
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Henry Koziel
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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Souvenir D. Tachado
*Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and
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  • FIGURE 1.
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    FIGURE 1.

    Restoration of TLR-4–mediated increases in MyD88-dependent TNF-α release in HIV+ AM by rapamycin. A, mTOR inhibition by rapamycin reversed blunted increases in TLR-4–mediated MyD88-dependent TNF-α release in HIV+ AMs. Healthy and HIV+ AMs were pretreated with 2.5 μg/ml rapamycin for 1 h, followed by incubation with 10 μg/ml LA for 24 h, and cell-free supernatant was analyzed for TNF-α by ELISA. Data shown are mean ± SEM of three independent experiments done in triplicate. *p < 0.01 compared with healthy with LA, **p < 0.05 compared with HIV+ in the absence of rapamycin. N.S., not significant compared with healthy AMs with LA alone. B, Rapamycin alone did not stimulate the release of TNF-α from healthy human AMs. Healthy AMs were treated with and without rapamycin for 24 h, and cell-free supernatant was analyzed for TNF-α by ELISA. Data shown are mean ± SEM of three independent experiments done in triplicate. C, TLR-4–mediated MyD88-independent IL-10 release was preserved in HIV+ AMs and inhibited by rapamycin. Healthy and HIV+ AMs were treated with 10 μg/ml LA in the presence or absence of 2.5 μg/ml rapamycin for 24 h, and cell-free supernatant was analyzed for IL-10 by ELISA. n = 3 subjects for each group. Data shown are mean ± SEM of three independent experiments done in triplicate. *p < 0.01 compared with both HIV+ and healthy AMs in the absence of rapamycin, **p < 0.05 compared with healthy AMs with LA alone.

  • FIGURE 2.
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    FIGURE 2.

    Dependence of LA-mediated TNF-α release in HIV+ macrophages on changes in mTOR activity. A, Western blot analysis of healthy and HIV+ AMs following inhibition of activated mTOR with rapamycin for 1 h and incubated with LA for 15 min. Membranes were probed with anti-p70s6K Ab, and immunoreactive bands were detected with HRP-conjugated secondary Ab. Total ERK1/2 was used to validate protein-loading equivalence after membrane stripping. Densitometric analysis is displayed beneath Western blots. The Western blot is a representative experiment of three independent experiments from three different individuals for each group with similar results (n = 3). *p = 0.01 compared with unstimulated healthy AMs, **p = 0.05 compared with HIV+ AM in the presence of LA alone. B, mTOR is constitutively active in HIV+ macrophages. Western blot analysis of U937 cells and U1 cells in the presence or absence of LA for 15 min. Membranes were probed with anti–phospho-mTOR Ab, and immunoreactive bands were detected with HRP-conjugated secondary Ab. Total mTOR was used to validate protein-loading equivalence after membrane stripping. Densitometric analysis is displayed beneath the Western blots. Western blot is a representative experiment of one of four independent experiments with similar results. *p = 0.01 compared with unstimulated U937 cells. C, Western blot analysis of human mTOR after gene silencing using siRNA. Total ERK1/2 was used to validate protein-loading equivalence after membrane stripping. A representative blot shows results from one of four independent experiments with similar results. Densitometric analysis is displayed under the blot. D–G, Human U937 and U1 macrophages were pretreated with either mTOR siRNA or scrambled (N.S.) siRNA and were then incubated with or without 10 μg/ml LA for 24 h; and the cell-free supernatant was analyzed for TNF-α release (D and E), *p = 0.05 compared with N.S siRNA with LA, or IL-10 (F and G), *p = 0.03 compared with N.S. siRNA with LA, **p = 0.05 compared with N.S. siRNA with LA by ELISA. Data shown are mean ± SEM of three independent experiments done in triplicate with similar results.

  • FIGURE 3.
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    FIGURE 3.

    Restoration by mTOR inhibition of MyD88-dependent signaling in human macrophages. A, MyD88/IRAK interaction restoration as a function of dose-dependent increases in rapamycin accounted for reversal of blunting effect of activated mTOR on LA-induced increases in TNF-α. U1 cells were preincubated in the presence of 2.5 μg/ml rapamycin for 1 h followed by LA for 15 min. Detergent soluble extracts were immunoprecipitated with anti-MyD88 Ab and immunoblotted with anti–p-IRAK Ab. Densitometric analysis for p-IRAK bands is displayed beneath the blot. Blot is representative of three independent experiments with similar results. *p = 0.05 compared with LA without rapamycin. B, Functional silencing of MyD88 led to increased phosphorylation of p70s6K in macrophages in response to LA. U937 cells were pretreated with shRNA MyD88 and N.S. shRNA. Cells were differentiated with phorbol ester, challenged with LA in either the presence or the absence of rapamycin. Total cell lysates were isolated and probed with anti–phospho-p70s6K Ab, and the immunoreactive band was detected with HRP-conjugated secondary Ab. β-actin expression was used to validate protein-loading equivalence after stripping membrane. Densitometric analysis is displayed beneath the Western blots. The Western blot is a representative experiment of four independent experiments with similar results. *p = 0.01 compared with MyD88−/− cells in the presence of LA. C and D, Inhibition of mTOR augmented LA-mediated TNF-α release in MyD88−/− macrophages, but IL-10 release was inhibited. U937 cells were pretreated with either shRNA MyD88 or N.S. shRNA, and differentiated with phorbol ester, challenged with LA in the presence or absence of rapamycin, and incubated for 24 h. Cell-free supernatant was assayed for TNF-α release (C) and IL-10 release (D) by ELISA. Results are mean ± SEM of three independent experiments performed in triplicate with similar results. #p < 0.01 compared with N.S. shRNA with LA, *p < 0.05 compared with shRNA MyD88 with LA (C). *p < 0.01 compared with both shRNAs with LA (D).

  • FIGURE 4.
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    FIGURE 4.

    ERK1/2 and JNK1/2 phosphorylation pattern altered by mTOR inhibition through declines in MKP-1 phosphorylation in HIV+ macrophages. HIV+ macrophages (U1 cells) were differentiated with phorbol ester and pretreated with 2.5 μg/ml rapamycin for 1 h, followed by LA incubation for different times. Cell lysates were analyzed by Milliplex Human MAP Kit panel. Results are representative of three independent experiments performed in duplicate. A, Phospho-ERK1/2; *p < 0.01 compared with LA alone for 15 min, **p < 0.005 compared with LA for 15 min. B, Phospho-JNK1/2; *p < 0.05 compared with unstimulated cells and compared with LA alone for 15 min. C, U1 cells were differentiated with phorbol ester and pretreated with 2.5 μg/ml rapamycin for 1 h followed by LA incubation for different times. Membranes were probed with anti–phospho-MKP-1 Ab, and immunoreactive bands were detected with HRP-conjugated secondary Ab. Total MKP-1 expression was used to validate protein-loading equivalence after membrane stripping. Densitometric analysis is displayed beneath the Western blots. The Western blot is a representative experiment of four independent experiments with similar results. D, U1 cells were differentiated with phorbol ester and challenged with LA (10 μg/ml), incubated for different times. Membranes were probed with anti–phospho-MKP-1 Ab, and immunoreactive bands were detected with HRP-conjugated secondary Ab. Total MKP-1 expression was used to monitor protein-loading equivalence after membrane stripping. The Western blot is a representative experiment of four independent experiments with similar results.

  • FIGURE 5.
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    FIGURE 5.

    Working model describing how in HIV+ macrophages constitutively activated PI3K elicits blunting of LA-induced increases in TNF-α release through sustained mTOR activation. The PI3K/Akt/mTOR signaling pathway is constitutively active in HIV+ macrophages and inhibits the expression of TNF-α by (1) direct mTOR-dependent inhibition of MyD88-dependent signaling pathway, (2) IL-10 production through the activation of mTOR, and (3) destabilization of a pan-specific MKP-1 expression through declines in its phosphorylation status. Inhibition of MKP-1 elicited negative feedback on ERK1/2, and JNK1/2 phosphorylation prolonged ERK1/2 and JNK1/2 activation by declines in its dephosphorylation.

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The Journal of Immunology: 187 (11)
The Journal of Immunology
Vol. 187, Issue 11
1 Dec 2011
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Mammalian Target of Rapamycin Inhibition in Macrophages of Asymptomatic HIV+ Persons Reverses the Decrease in TLR-4–Mediated TNF-α Release through Prolongation of MAPK Pathway Activation
Xin Li, Xinbing Han, Juliana Llano, Medhavi Bole, Xiuqin Zhou, Katharine Swan, Asha Anandaiah, Benjamin Nelson, Naimish R. Patel, Peter S. Reinach, Henry Koziel, Souvenir D. Tachado
The Journal of Immunology December 1, 2011, 187 (11) 6052-6058; DOI: 10.4049/jimmunol.1101532

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Mammalian Target of Rapamycin Inhibition in Macrophages of Asymptomatic HIV+ Persons Reverses the Decrease in TLR-4–Mediated TNF-α Release through Prolongation of MAPK Pathway Activation
Xin Li, Xinbing Han, Juliana Llano, Medhavi Bole, Xiuqin Zhou, Katharine Swan, Asha Anandaiah, Benjamin Nelson, Naimish R. Patel, Peter S. Reinach, Henry Koziel, Souvenir D. Tachado
The Journal of Immunology December 1, 2011, 187 (11) 6052-6058; DOI: 10.4049/jimmunol.1101532
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