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Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes.

ABSTRACT: Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell-specific deletion of dynamin 2, an essential component of endocytosis, resulted in reduced TCR signaling strength, impaired homeostatic proliferation, and the inability to undergo clonal expansion in vivo. Blocking endocytosis resulted in a failure to maintain mammalian target of rapamycin (mTOR) activity and to stably induce the transcription factor myelocytomatosis oncogene (c-Myc), which led to metabolic stress and a defect in cell growth. Our results support the concept that the TCR can continue to signal after it is internalized from the cell surface, thereby enabling sustained signaling and cell proliferation.

SUBMITTER: Willinger T

PROVIDER: S-EPMC4394306 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes.

Willinger Tim T Staron Matthew M Ferguson Shawn M SM De Camilli Pietro P Flavell Richard A RA

Proceedings of the National Academy of Sciences of the United States of America 20150323 14

Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell-specific deletion of dynamin 2, an essential component of ...[more]

PMID: 25831514

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Project description:BACKGROUND:Lymphotoxin ? receptor (LT?R) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-?B and AP-1 pathways. Yet, the intracellular distribution of LT?R, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LT?R internalization to its signaling potential. METHODS:Intracellular localization of LT?R in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LT?R-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-?B pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LT?R stimulation was assessed by qRT-PCR analysis. RESULTS:We demonstrated that LT?R was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LT?R internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-?B signaling represented by increased degradation of I?B? inhibitor and elevated expression of LT?R target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LT?R-triggered activation of the non-canonical branch of the NF-?B pathway. CONCLUSIONS:Our work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LT?R stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Video Abstract.

Project description:RATIONALE:A hallmark of chronic inflammatory disorders is persistence of proinflammatory macrophages in diseased tissues. In atherosclerosis, this is associated with dyslipidemia and oxidative stress, but mechanisms linking these phenomena to macrophage activation remain incompletely understood. OBJECTIVE:To investigate mechanisms linking dyslipidemia, oxidative stress, and macrophage activation through modulation of immunometabolism and to explore therapeutic potential targeting specific metabolic pathways. METHODS AND RESULTS:Using a combination of biochemical, immunologic, and ex vivo cell metabolic studies, we report that CD36 mediates a mitochondrial metabolic switch from oxidative phosphorylation to superoxide production in response to its ligand, oxidized LDL (low-density lipoprotein). Mitochondrial-specific inhibition of superoxide inhibited oxidized LDL-induced NF-?B (nuclear factor-?B) activation and inflammatory cytokine generation. RNA sequencing, flow cytometry, 3H-labeled palmitic acid uptake, lipidomic analysis, confocal and electron microscopy imaging, and functional energetics revealed that oxidized LDL upregulated effectors of long-chain fatty acid uptake and mitochondrial import, while downregulating fatty acid oxidation and inhibiting ATP5A (ATP synthase F1 subunit alpha)-an electron transport chain component. The combined effect is long-chain fatty acid accumulation, alteration of mitochondrial structure and function, repurposing of the electron transport chain to superoxide production, and NF-?B activation. Apoe null mice challenged with high-fat diet showed similar metabolic changes in circulating Ly6C+ monocytes and peritoneal macrophages, along with increased CD36 expression. Moreover, mitochondrial reactive oxygen species were positively correlated with CD36 expression in aortic lesional macrophages. CONCLUSIONS:These findings reveal that oxidized LDL/CD36 signaling in macrophages links dysregulated fatty acid metabolism to oxidative stress from the mitochondria, which drives chronic inflammation. Thus, targeting to CD36 and its downstream effectors may serve as potential new strategies against chronic inflammatory diseases such as atherosclerosis.

Dataset Information

Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes.

Publications

Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes.

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