Rapamycin‐sensitive signals control TCR/CD28‐driven Ifng, Il4 and Foxp3 transcription and promoter region methylation

Abstract

The mammalian target of rapamycin (mTOR) controls T-cell differentiation in response to polarizing cytokines. We previously found that mTOR blockade by rapamycin (RAPA) delays the G1-S cell cycle transition and lymphocyte proliferation. Here, we report that both mTOR complex 1 and mTOR complex 2 are readily activated following TCR/CD28 engagement and are critical for early expression of Ifng, Il4 and Foxp3, and for effector T cell differentiation in the absence of polarizing cytokines. While inhibition of mTOR complex 1 and cell division were evident at low doses of RAPA, inhibition of mTOR complex 2, Ifng, Il4 and Foxp3 expression, and T-cell polarization required higher doses and more prolonged treatments. We found that while T-bet and GATA3 were readily induced following TCR/CD28 engagement, administration of RAPA delayed their expression, and interfered with the loss of DNA methylation within Ifng and Il4 promoter regions. In contrast, RAPA prevented activation-dependent DNA methylation of the Foxp3 promoter favoring Foxp3 expression. As a result, RAPA-cultured cells lacked immediate effector functions and instead were enriched for IL-2 1 cells. We propose that mTOR-signaling, by timing the expression of critical transcription factors and DNA methylation of proximal promoter regions, regulates transcriptional competence at immunologically relevant sites and hence lymphocyte differentiation. 2086Introduction TOR (target of rapamycin) integrates environmental cues, including amino acid and nutrient availability, energy stores and growth factor signaling, and subsequently directs cell growth and proliferation in yeast and mammals. Mammalian (m)TOR exists in two complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [1]. mTORC1 comprises mTOR, Raptor, mLST8 and PRAS40 while the functionally distinct mTORC2 contains mTOR, mLST8 and the unique regulatory proteins Rictor, mSIN1 and PROTOR (reviewed in [2]). mTORC1 controls transcription and translation in response to nutrient and amino acid levels, growth factors and cytokines through phosphorylation of the p70 S6 kinase (p70S6K) and the initiation factor 4E-binding protein 1 (4EBP1), and is strongly sensitive to inhibition by the naturally occurring compound rapamycin (RAPA). While the function of mTORC2 remains largely uncharacterized, it has been shown to regulate aspects of the actin cytoskeleton, and to directly phosphorylate Akt (Ser473), PKC-a (Ser657) and SGK1 (Ser422). While mTORC2 is insensitive to transient RAPA administration, prolonged RAPA treatment hinders mTORC2 assembly, thus blocking mTORC2 function in some cells [2].In T cells, mTOR appears to regulate several aspects of lymphocyte biology and to lie at the crossroads of T-cell proliferation and tolerance [3,4]. T-cell-specific deletion of Frap1 (encoding for mTOR) hinders Th1 and Th2 differentiation in response to polarizing cytokines favoring differentiation of Foxp3 1 cells [5]. In contrast, deletion of Rictor abrogates Th1and Th2 cell differentiation without, however, diverting...

Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases

“…Diminished T REG -cell frequency has been linked to mTORC1-sensitive methylation of the Foxp3 promoter 112 , whereas rapamycin treatment reduced the production of IL-4 by DN T cells in vivo 109 , accounting for an increased production of anti-DNA autoantibodies by B cells 113,114 . The benefits of rapamycin can also be attributed to T REG -cell expansion 109 via activation of mTORC2, at least in patients with SLE 106 .…”

Oxidative stress in the pathology and treatment of systemic lupus erythematosus

“…74 FoxP3, conversely, is an anti-inflammatory transcription factor whose expression in mouse regulatory T (T REG ) cells is maintained by serine/threonine-protein kinase mTOR-sensitive methylation of the Foxp3 promoter, and is thus diminished in the absence of methylation. 75 …”

“…7 Although a dose of 1.2 g per day was ineffective, NAC was found to be safe, and doses of 2.4 g per day and 4.8 g per day were effective in reversing glutathione depletion and improving disease activity and fatigue. Blocking activation of mTOR and promoting expansion of CD4 + CD25 + FOXP3 + T-cell subsets, possibly through preventing T-cell activation-induced methylation and silencing of the FOXP3 promoter, 75 NAC treatment was able to inhibit anti-DNA antibody production. 7 Indeed, NAC reversed the expansion of CD4 − CD8 − T cells, which exhibited the most prominent mTOR activation before treatment with NAC 7 and had been deemed responsible for promoting anti-DNA autoantibody production by B cells.…”

mTOR and lymphocyte metabolism

“…Moreover, mTOR signaling influences epigenetic modifications that accompany T cell differentiation. Rapamycin treatment during T cell activation leads to increased CpG methylation at promoter regions of Il4 and Ifng [60], but reduces CpG methylation and increases permissive histone methylation at Foxp3 promoter region[30,60]. Interestingly, epigenetic regulation is likely an evolutionarily conserved function of mTOR, as TOR signaling in yeast is linked to histone acetylation[61].…”