Abstract
In order to establish infection, intra-macrophage parasite Leishmania donovani needs to inhibit host defense parameters like inflammatory cytokine production and apoptosis. In the present study, we demonstrate that the parasite achieves both by exploiting a single host regulator AKT for modulating its downstream transcription factors, β-catenin and FOXO-1. L. donovani-infected RAW264.7 and bone marrow-derived macrophages (BMDM) treated with AKT inhibitor or dominant negative AKT constructs showed decreased anti-inflammatory cytokine production and increased host cell apoptosis resulting in reduced parasite survival. Infection-induced activated AKT triggered phosphorylation-mediated deactivation of its downstream target, GSK-3β. Inactivated GSK-3β, in turn, could no longer sequester cytosolic β-catenin, an anti-apoptotic transcriptional regulator, as evidenced from its nuclear translocation during infection. Constitutively active GSK-3β-transfected L. donovani-infected cells mimicked the effects of AKT inhibition and siRNA-mediated silencing of β-catenin led to disruption of mitochondrial potential along with increased caspase-3 activity and IL-12 production leading to decreased parasite survival. In addition to activating anti-apoptotic β-catenin, phospho-AKT inhibits activation of FOXO-1, a pro-apoptotic transcriptional regulator. Nuclear retention of FOXO-1, inhibited during infection, was reversed when infected cells were transfected with dominant negative AKT constructs. Overexpression of FOXO-1 in infected macrophages not only documented increased apoptosis but promoted enhanced TLR4 expression and NF-κB activity along with an increase in IL-1β and decrease in IL-10 secretion. In vivo administration of AKT inhibitor significantly decreased liver and spleen parasite burden and switched cytokine balance in favor of host. In contrast, GSK-3β inhibitor did not result in any significant change in infectivity parameters. Collectively our findings revealed that L. donovani triggered AKT activation to regulate GSK-3β/β-catenin/FOXO-1 axis, thus ensuring inhibition of both host cell apoptosis and immune response essential for its intra-macrophage survival.
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Abbreviations
- DN:
-
dominant negative
- WT:
-
wild type
- CA:
-
constituitively active
- BMDM:
-
bone marrow-derived macrophages
- HA:
-
haemagglutinin
- LDU:
-
Leishman–Donovan Units
- GFP:
-
green fluorescent protein
- TR:
-
texas red
- DAPI:
-
4′,6-diamidino-2-phenylindole
- DC:
-
dendritic cells
- Ser:
-
serine
- Thr:
-
threonine
- PTEN:
-
phosphatase and tensin homolog
- DiOC6(3):
-
3,3'-dihexyloxacarbocyanine iodide
References
Bhardwaj S, Srivastava N, Sudan R, Saha B . Leishmania interferes with host cell signaling to devise a survival strategy. J Biomed Biotechnol 2010; 2010: 109189.
Cecilio P, Pérez-Cabezas B, Santarém N, Maciel J, Rodrigues V, Cordeiro da Silva A et al. Deception and manipulation: the arms of Leishmania, a successful parasite. Front Immunol 2014; 5: 480.
Zhang Y, Wang X, Yang H, Liu H, Lu Y, Han L et al. Kinase AKT controls innate immune cell development and function. Immunology 2013; 140: 143–152.
Neves BM, Silvestre R, Resende M, Ouaissi A, Cunha J, Tavares J et al. Activation of phosphatidylinositol 3-kinase/Akt and impairment of nuclear factor-kappaB: molecular mechanisms behind the arrested maturation/activation state of Leishmania infantum-infected dendritic cells. Am J Pathol 2010; 177: 2898–2911.
Ruhland A, Leal N, Kima PE . Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis. Cell Microbiol 2007; 9: 84–96.
Faherty CS, Maurelli AT . Staying alive: bacterial inhibition of apoptosis during infection. Trends Microbiol 2008; 16: 173–180.
Chuenkova MV, PereiraPerrin M . Trypanosoma cruzi targets Akt in host cells as an intracellular antiapoptotic strategy. Sci Signal 2009; 2: ra74.
Thi EP, Lambertz U, Reiner NE . Sleeping with the enemy: how intracellular pathogens cope with a macrophage lifestyle. PLoS Pathog 2012; 8: e1002551.
Antoniv TT, Ivashkiv LB . Interleukin-10-induced gene expression and suppressive function are selectively modulated by the PI3K-Akt-GSK3 pathway. Immunology 2011; 132: 567–577.
Guha M, Mackman N . The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells. J Biol Chem 2002; 277: 32124–32132.
Fang D, Hawke D, Zheng Y, Xia Y, Meisenhelder J, Nika H et al. Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J Biol Chem 2007; 282: 11221–11229.
Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA . Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995; 378: 785–789.
Monick MM, Carter AB, Robeff PK, Flaherty DM, Peterson MW, Hunninghake GW . Lipopolysaccharide activates Akt in human alveolar macrophages resulting in nuclear accumulation and transcriptional activity of beta-catenin. J Immunol 2001; 166: 4713–4720.
He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT et al. Identification of c-MYC as a target of the APC pathway. Science 1998; 281: 1509–1512.
Yuan L, Choi K, Khosla C, Zheng X, Higashikubo R, Chicoine MR et al. Tissue transglutaminase 2 inhibition promotes cell death and chemosensitivity in glioblastomas. Mol Cancer Ther 2005; 4: 1293–1302.
Kaga S, Zhan L, Altaf E, Maulik N . Glycogen synthase kinase-3beta/beta-catenin promotes angiogenic and anti-apoptotic signaling through the induction of VEGF, Bcl-2 and survivin expression in rat ischemic preconditioned myocardium. J Mol Cell Cardiol 2006; 40: 138–147.
Tzivion G, Dobson M, Ramakrishnan G . FoxO transcription factors; Regulation by AKT and 14-3-3 proteins. Biochim Biophys Acta 2011; 1813: 1938–1945.
Wang Y, Zhou Y, Graves DT . FOXO transcription factors: their clinical significance and regulation. Biomed Res Int 2014; 2014: 925350.
Fan W, Morinaga H, Kim JJ, Bae E, Spann NJ, Heinz S et al. FoxO1 regulates Tlr4 inflammatory pathway signalling in macrophages. EMBO J 2010; 29: 4223–4236.
Seiler F, Hellberg J, Lepper PM, Kamyschnikow A, Herr C, Bischoff M et al. FOXO transcription factors regulate innate immune mechanisms in respiratory epithelial cells. J Immunol 2013; 190: 1603–1613.
Duan Y, Liao AP, Kuppireddi S, Ye Z, Ciancio MJ, Sun J . beta-Catenin activity negatively regulates bacteria-induced inflammation. Lab Invest 2007; 87: 613–624.
Beurel E, Jope RS . The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways. Prog Neurobiol 2006; 79: 173–189.
Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford WL, Bolon B et al. Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration. Science 2000; 287: 1040–1046.
Lawrence T . The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol 2009; 1: a001651.
Huang SM, Mishina YM, Liu S, Cheung A, Stegmeier F, Michaud GA et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 2009; 461: 614–620.
Haq S, Michael A, Andreucci M, Bhattacharya K, Dotto P, Walters B et al. Stabilization of beta-catenin by a Wnt-independent mechanism regulates cardiomyocyte growth. Proc Natl Acad Sci USA 2003; 100: 4610–4615.
Sakanaka C . Phosphorylation and regulation of beta-catenin by casein kinase I epsilon. J Biochem 2002; 132: 697–703.
Kamo N, Ke B, Busuttil RW, Kupiec-Weglinski JW . PTEN-mediated Akt/beta-catenin/Foxo1 signaling regulates innate immune responses in mouse liver ischemia/reperfusion injury. Hepatology 2013; 57: 289–298.
Qiang L, Banks AS, Accili D . Uncoupling of acetylation from phosphorylation regulates FoxO1 function independent of its subcellular localization. J Biol Chem 2010; 285: 27396–27401.
Vazquez-Lopez R, Argueta-Donohué J, Wilkins-Rodríguez A, Escalona-Montaño A, Aguirre-García M, Gutiérrez-Kobeh L . Leishmania mexicana amastigotes inhibit p38 and JNK and activate PI3K/AKT: role in the inhibition of apoptosis of dendritic cells. Parasite Immunol 2015; 37: 579–589.
Nandan D, Camargo de Oliveira C, Moeenrezakhanlou A, Lopez M, Silverman JM, Subek J et al. Myeloid cell IL-10 production in response to Leishmania involves inactivation of glycogen synthase kinase-3beta downstream of phosphatidylinositol-3 kinase. J Immunol 2012; 188: 367–378.
Jacobs KM, Bhave SR, Ferraro DJ, Jaboin JJ, Hallahan DE, Thotala D . GSK-3beta: A Bifunctional Role in Cell Death Pathways. Int J Cell Biol 2012; 2012: 930710.
Manning BD, Cantley LC . AKT/PKB signaling: navigating downstream. Cell 2007; 129: 1261–1274.
Orr SJ, Burg AR, Chan T, Quigley L, Jones GW, Ford JW et al. LAB/NTAL facilitates fungal/PAMP-induced IL-12 and IFN-gamma production by repressing beta-catenin activation in dendritic cells. PLoS Pathog 2013; 9: e1003357.
Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO et al. The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 2004; 14: 395–403.
Hoogeboom D, Essers MA, Polderman PE, Voets E, Smits LM, Burgering BM . Interaction of FOXO with beta-catenin inhibits beta-catenin/T cell factor activity. J Biol Chem 2008; 283: 9224–9230.
Fukao T, Koyasu S . PI3K and negative regulation of TLR signaling. Trends Immunol 2003; 24: 358–363.
Gupta P, Giri J, Srivastav S, Chande AG, Mukhopadhyaya R, Das PK et al. Leishmania donovani targets tumor necrosis factor receptor-associated factor (TRAF) 3 for impairing TLR4-mediated host response. FASEB J 2014; 28: 1756–1768.
Srivastav S, Kar S, Chande AG, Mukhopadhyaya R, Das PK . Leishmania donovani exploits host deubiquitinating enzyme A20, a negative regulator of TLR signaling, to subvert host immune response. J Immunol 2012; 189: 924–934.
Kar S, Ukil A, Sharma G, Das PK . MAPK-directed phosphatases preferentially regulate pro- and anti-inflammatory cytokines in experimental visceral leishmaniasis: involvement of distinct protein kinase C isoforms. J Leukoc Biol 2010; 88: 9–20.
Murray HW, Miralles GD, Stoeckle MY, McDermott DF . Role and effect of IL-2 in experimental visceral leishmaniasis. J Immunol 1993; 151: 929–938.
Kuijl C, Savage ND, Marsman M, Tuin AW, Janssen L, Egan DA et al. Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1. Nature 2007; 450: 725–730.
He X, Saint-Jeannet JP, Woodgett JR, Varmus HE, Dawid IB . Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature 1995; 374: 617–622.
Stambolic V, Woodgett JR . Mitogen inactivation of glycogen synthase kinase-3 beta in intact cells via serine 9 phosphorylation. Biochem J 1994; 303: 701–704.
Frescas D, Valenti L, Accili D . Nuclear trapping of the forkhead transcription factor FoxO1 via Sirt-dependent deacetylation promotes expression of glucogenetic genes. J Biol Chem 2005; 280: 20589–20595.
Acknowledgements
This work was supported by the Department of Science and Technology (SB/SO/BB-0055/2013, SERB/F/4467/2013-14), SERB EMR/2014/000287, University Grants Commission (F. No.6-10/2016(IC)), Government of West Bengal, Department of Biotechnology, (221/BT(Estt)/RD-40/2014) and National Academy of Sciences, India (NASI). Fellowship was provided by Council of Scientific & Industrial Research (CSIR) and University Grants Commission (UGC). We thank DBT-CU-IPLS Core Facility for confocal microscope facility and CU-BD-COE for providing imaging and cytometry facility.
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Gupta, P., Srivastav, S., Saha, S. et al. Leishmania donovani inhibits macrophage apoptosis and pro-inflammatory response through AKT-mediated regulation of β-catenin and FOXO-1. Cell Death Differ 23, 1815–1826 (2016). https://doi.org/10.1038/cdd.2016.101
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DOI: https://doi.org/10.1038/cdd.2016.101
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