Mediators Inflamm. 2016;2016:9605253. doi: 10.1155/2016/9605253.
PGC-1α-dependent mitochondrial adaptation is necessary to sustain IL-2-induced activities in human NK cells
Dante Miranda2, Claudia Jara1, Jorge Ibañez1, Viviana Ahumada1, Claudio Acuña-Castillo1, Adrian Martin1, Alexandra Córdova1, Margarita Montoya1.
1 Department of Biology, Faculty of Chemistry and Biology, University of Santiago of Chile, Av. Libertador Bernardo O’Higgins 3363, 9170022 Santiago, Chile.
2 Department of Biochemistry and Molecular Biology, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Sergio Livingstone 1007, Independencia, Santiago, Chile.
Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass as well as the mitochondrial membrane potential increased in a PGC-1α dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.
Natural killer (NK) cells are a specialized heterogeneous population of lymphocytes that belong to the family of group 1 innate lymphocytes (ILC1). They contribute to host antimicrobial and antitumor defense reactions through their ability to exert a cytotoxicity activity against target cells and by secreting major inflammatory cytokines, such as IFN-γ which plays a key role in controlling infection. Thus, NK cells act as regulatory cells in the immune system, influencing other cells and responses, and acting as a link between the adaptive and innate immune response [1-3].
IL-2 is a pluripotent cytokine predominantly produced by T cells that can activate NK cells [4, 5], promote their migration within target tissues , and increase the secretion of cytokines and other small molecules. Upon stimulation with IL-2, NK cells develop a strong cytolytic activity against target cells, named LAK activity by increasing the number and size of cytoplasmic granules , augmenting the expression of effector molecules , and altering the surface expression of receptors . Moreover, it has been described that IL-2 has the potential to restore the cytotoxicity and granular content of exhausted NK cells . In this context, IL-2 has been used as an immunotherapeutic agent to promote NK cell antitumor activity [11, 12]. In addition, IL-2 has been shown to be critical in viral and bacterial defenses as well in vaccine protection. Accordingly, we thought that to execute this effector function, NK cells should require a large amount of energy.
Recently, it has been shown that metabolism regulates the acquisition of effector functions in different immune cells. It is known, for example, that effector T cells increase glycolysis to support growth and proliferation [13, 14], but also to support the ability to produce IFN-γ . By contrast, naïve memory T cells and Treg increase mitochondrial metabolism for ATP synthesis [13, 14, 16]. Nonetheless, less is known about metabolism adaptations for NK cell functions, although it has been reported that mitochondrial dynamics are important for NK cell activity.
Thus, we decided to determine if human NK cells with LAK activity alter their metabolism to support the bioenergetic demand for performing cytotoxic activity and IFN-γ secretion more efficiently.
It is interesting to highlight that the results reported in the present study have been obtained using human isolated NK cells which provide direct insight over metabolic adaptation in human NK cells, field which remain poorly characterized.
Figure 1. Human NK cells in contact with Daudi target cells. Human NK cells were purified and incubated with MitoTracker Red FM probe and Daudi cells were culture and incubated with CellTracker Green. Then, both types of cells were co-incubated and soon afterwards were analyzed by fluorescence microscopy.
Our results showed that LAK activity was established at 48 h of IL-2 incubation with no further increase. As we expected, mitochondrial mass and mitochondrial potential were significantly increased, however these changes occurred after 72 h of IL-2 incubation. These results suggest that mitochondrial ATP synthesis could be important in supporting, instead of developing, the LAK phenotype. These observation prompted us to analyze mitochondrial biogenesis. Given that PCG-1α is required for maintaining mitochondrial homeostasis we decided to investigate the influence of PGC-1α expression on IL-2-mediated mitochondrial mass and potential increase.
We discovered that the IL-2 increase in both mitochondrial mass and mitochondrial potential in human NK cells, was dependent on the increased expression of PGC-1 transcriptional co-factor.
Moreover, the inhibition of PGC-1α mRNA expression resulted in an inhibition of the IL-2-mediated increase in mitochondrial mass and mitochondrial potential. Also, we observed that inhibition of mitochondrial ATP synthesis inhibited IL-2 activated ACNK. These results suggest an important role that mitochondria are playing in IL-2 induced LAK activity in human HPNK cells.
In addition, given that NK cells act as regulatory cells in the immune system principally by secreting IFN-γ, we also decide to investigate if these changes in mitochondrial adaptations under IL-2 stimulation through increasing PGC-1α mRNA expression were necessary for IFN-γ secretion.
As we expected, we demonstrated that the optimum secretion of IFN-γ induced by IL-2, was dependent on the increased expression of PGC-1α. This result turn out to be particularly interesting, since IFN-γ seem to be crucial to mount an efficient immune response against infections, tumor cells or vaccination.
Thus, the main finding of this work was to demonstrate that IL-2 induces an increase in mitochondrial mass as well as membrane potential in human NK cells in a manner dependent on an increase of PGC-1α expression. Our work shows that mitochondrial dynamics are essential for supporting IL-2-activated effector functions such as LAK activity and IFN-γ secretion which in turn play a key role in the development of an effective immune response against pathogens by human NK cells.
Figure 2. Possible mechanisms involved in PGC-1α-dependent mitochondrial adaptation in IL-2-induced activities in human NK cells.
1. Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol. 1999;17:189-220. doi: 10.1146/annurev.immunol.17.1.189. PubMed PMID: 10358757.
2. Orange JS, Ballas ZK. Natural killer cells in human health and disease. Clin Immunol. 2006;118(1):1-10. doi: 10.1016/j.clim.2005.10.011. PubMed PMID: 16337194.
3. Paolini R, Bernardini G, Molfetta R, Santoni A. NK cells and interferons. Cytokine Growth Factor Rev. 2015;26(2):113-20. doi: 10.1016/j.cytogfr.2014.11.003. PubMed PMID: 25443799.
4. Bonnema JD, Rivlin KA, Ting AT, Schoon RA, Abraham RT, Leibson PJ. Cytokine-enhanced NK cell-mediated cytotoxicity. Positive modulatory effects of IL-2 and IL-12 on stimulus-dependent granule exocytosis. J Immunol. 1994;152(5):2098-104. PubMed PMID: 7907631.
5. Phillips JH, Gemlo BT, Myers WW, Rayner AA, Lanier LL. In vivo and in vitro activation of natural killer cells in advanced cancer patients undergoing combined recombinant interleukin-2 and LAK cell therapy. J Clin Oncol. 1987;5(12):1933-41. PubMed PMID: 3500280.
6. Hagenaars M, Zwaveling S, Kuppen PJ, Ensink NG, Eggermont AM, Hokland ME, et al. Characteristics of tumor infiltration by adoptively transferred and endogenous natural-killer cells in a syngeneic rat model: implications for the mechanism behind anti-tumor responses. Int J Cancer. 1998;78(6):783-9. PubMed PMID: 9833773.
7. Trinchieri G, Matsumoto-Kobayashi M, Clark SC, Seehra J, London L, Perussia B. Response of resting human peripheral blood natural killer cells to interleukin 2. J Exp Med. 1984;160(4):1147-69. PubMed PMID: 6434688; PubMed Central PMCID: PMCPMC2187474.
8. Zhou J, Zhang J, Lichtenheld MG, Meadows GG. A role for NF-kappa B activation in perforin expression of NK cells upon IL-2 receptor signaling. J Immunol. 2002;169(3):1319-25. PubMed PMID: 12133954.
9. Chrul S, Polakowska E, Szadkowska A, Bodalski J. Influence of interleukin IL-2 and IL-12 + IL-18 on surface expression of immunoglobulin-like receptors KIR2DL1, KIR2DL2, and KIR3DL2 in natural killer cells. Mediators Inflamm. 2006;2006(4):46957. doi: 10.1155/MI/2006/46957. PubMed PMID: 17047292; PubMed Central PMCID: PMCPMC1618942.
10. Bhat R, Watzl C. Serial killing of tumor cells by human natural killer cells–enhancement by therapeutic antibodies. PLoS One. 2007;2(3):e326. doi: 10.1371/journal.pone.0000326. PubMed PMID: 17389917; PubMed Central PMCID: PMCPMC1828617.
11. Moretta L, Pietra G, Montaldo E, Vacca P, Pende D, Falco M, et al. Human NK cells: from surface receptors to the therapy of leukemias and solid tumors. Front Immunol. 2014;5:87. doi: 10.3389/fimmu.2014.00087. PubMed PMID: 24639677; PubMed Central PMCID: PMCPMC3945935.
12. Waldmann TA. The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat Rev Immunol. 2006;6(8):595-601. doi: 10.1038/nri1901. PubMed PMID: 16868550.
13. Gerriets VA, Rathmell JC. Metabolic pathways in T cell fate and function. Trends Immunol. 2012;33(4):168-73. doi: 10.1016/j.it.2012.01.010. PubMed PMID: 22342741; PubMed Central PMCID: PMCPMC3319512.
14. Pearce EL, Pearce EJ. Metabolic pathways in immune cell activation and quiescence. Immunity. 2013;38(4):633-43. doi: 10.1016/j.immuni.2013.04.005. PubMed PMID: 23601682; PubMed Central PMCID: PMCPMC3654249.
15. Chang CH, Curtis JD, Maggi LB, Jr., Faubert B, Villarino AV, O’Sullivan D, et al. Posttranscriptional control of T cell effector function by aerobic glycolysis. Cell. 2013;153(6):1239-51. doi: 10.1016/j.cell.2013.05.016. PubMed PMID: 23746840; PubMed Central PMCID: PMCPMC3804311.
16. Pollizzi KN, Powell JD. Integrating canonical and metabolic signalling programmes in the regulation of T cell responses. Nat Rev Immunol. 2014;14(7):435-46. doi: 10.1038/nri3701. PubMed PMID: 24962260; PubMed Central PMCID: PMCPMC4390057.