mTOR and GSK-3 shape the CD4+ T-cell stimulatory and differentiation capacity of myeloid DCs after exposure to LPS

Abstract

Prolonged inhibition of the kinase, mammalian target of rapamycin (mTOR), during myeloid dendritic cell (DC) generation confers resistance to maturation. Recently, however, mTOR inhibition immediately before Toll-like receptor ligation has been found to exert proinflammatory effects on myeloid cells, notably enhanced IL-12p40/p70 production. We show, for the first time, that mouse or human DCs generated under mTOR inhibition exhibit markedly enhanced IL-12p70 production after lipopolysaccharide (LPS) stimulation, despite impaired costimulatory molecule expression and poor T-cell stimulatory ability. Consistent with this finding, we reveal that increased IL-12p40 production occurs predominantly in CD86 lo immature DCs. High IL-12p40/p70 production by CD86 lo DC resulted from failed down-regulation of glycogen synthase kinase-3 (GSK-3) activity and could not be ascribed to enhanced Akt function. Despite high IL-12p70 secretion, rapamycinconditioned, LPS-stimulated DCs remained poor T-cell stimulators, failing to enhance allogeneic Th1 cell responses. We also report that inhibition of GSK-3 impedes the ability of LPS-stimulated DCs to induce forkhead box p3 in CD4 ؉ CD25 ؊ T cells, as does the absence of IL-12p40/ p70. Thus, GSK-3 activity in DC is regulated via signaling linked to mTOR and modulates their capacity both to produce IL-12p40/p70 and induce forkhead box p3 in CD4 ؉ IntroductionMammalian target of rapamycin (mTOR) is an integrative kinase that coordinates environmental signals, especially those activating phosphoinositide 3-kinase (PI3K) and its effector, the Akt kinase. 1,2 The relationship between the 2 identified mTOR-containing complexes (mTORC1 and mTORC2) and PI3K/Akt is under intensive investigation, but it is understood that mTORC1 is located downstream of PI3K and activated by Akt. 1 Akt, however, lies both upstream and downstream of mTOR and must be phosphorylated on S473 by mTORC2 to be fully activated. 1 Although the immunosuppressant rapamycin (RAPA) potently targets mTORC1 activity to limit cell growth and proliferation, mTORC2 is RAPA-resistant, although prolonged RAPA exposure can limit its activity in some cells and tissues. 3 Consistent with ubiquitous leukocyte mTOR expression, RAPA exerts significant immunomodulatory effects. 4 At clinically relevant concentrations, it inhibits cytokine-induced proliferation of effector T cells while sparing the proliferation and function of regulatory T cells (Treg). [4][5][6] Both in vitro and in vivo, continued exposure to RAPA suppresses myeloid (m) dendritic cell (DC) generation, maturation, and T-cell stimulatory function. [7][8][9][10][11][12][13] More precisely, propagation of murine bone marrow (BM)-derived mDCs in RAPA (RAPA-conditioned mDCs; RAPA-DCs) generates mDCs with low surface major histocompatibility complex and costimulatory molecules, even after exposure to potent inflammatory stimuli, such as Toll-like receptor (TLR) ligands, and CD40 ligation. 8,[10][11][12] Although in vitro-generated RAPA-DCs are weak stimulators of T cells 8...

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“…MTORC2 also improves glycolytic metabolism by activating the AKT [121], [122]. Thus mTOR negatively regulates the GSK-3 activity to limit production of IL-12 [114], [117]. Our data is consistent with mTORC1 acting as negative regulator of IL-12 and facilitator of production of IL-10 after exposure to microbial agents.…”

Langerhans cell homeostasis in mice is dependent on mTORC1 but not mTORC2 function

“…46,47 We found GSK3␤ Ser9 phosphorylation reduced in ric ⌬/⌬ but not in rap ⌬/⌬ BM-DC. We propose that the effect of rapamycin on GSK-3 Ser9 phosphorylation is because of the simultaneous inhibition of both mTORC1 and mTORC2 function.…”

Murine dendritic cell rapamycin-resistant and rictor-independent mTOR controls IL-10, B7-H1, and regulatory T-cell induction

“…DCs were generated from BM cells, as described 19,25 using recombinant (r) mouse granulocyte macrophage-colony-stimulating factor and r mouse IL-4 (both 1000 U/mL; R&D Systems). On day 8 of culture, myeloid DCs were selected from nonadherent cells by anti-CD11c immunomagnetic bead purification (Miltenyi Biotec).…”

“…2,8,15 Conversely, RAPA has paradoxical, proinflammatory effects on DCs, including increased secretion of IL12p70 and IL-1b, with concomitant reduced secretion of IL-10 and expression of B7-H1. [16][17][18][19][20][21] These effects on DCs are mediated by augmentation of nuclear factor kB (NF-kB) activity and reduction in signal transducer and activator of transcription (STAT) 3 activity. 17,20,21 RAPA-insensitive mTORC2 regulates the actin cytoskeleton in nonimmune cells, 10,11 and insight is emerging into its function in T lymphocytes.…”