title: Loss of Nrf2 in bone marrow-derived macrophages impairs antigen-driven CD8(+) T cell function by limiting GSH and Cys availability.
abstract: NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8(+) T cell activation by using an antigen-driven coculture model consisting of Nrf2(-/-) and Nrf2(+/+) bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8(+) T cells. OT-1 CD8(+) T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257-264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257-264)-pulsed Nrf2(-/-) BMDMΦ with transgenic OT-1 CD8(+) T cells attenuated CD8(+) T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8(+) T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2(-/-) cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2(-/-) cocultures via addition of glutathione ester, N-acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2(+/+) BMDMΦ mimicked the effect of Nrf2(-/-) BMDMΦ on CD8(+) T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2(-/-) BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8(+) T cell function.
authors: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Lisa K
authors: LK
authors: Sha
authors: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Weixiao
authors: W
authors: Sha
authors: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Laura
authors: L
authors: Kuchler
authors: Department of Medicine II, University Medical Center, Johannes Gutenberg-University Mainz, 55116 Mainz, Germany.
authors: Andreas
authors: A
authors: Daiber
authors: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Annika K
authors: AK
authors: Giegerich
authors: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Andreas
authors: A
authors: Weigert
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine & Pharmacology TMP, and Goethe-University Frankfurt, 60596 Frankfurt am Main, Germany.
authors: Tilo
authors: T
authors: Knape
authors: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Ryan
authors: R
authors: Snodgrass
authors: Institute of Cardiovascular Physiology-Physiology I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Katrin
authors: K
authors: Schröder
authors: Institute of Cardiovascular Physiology-Physiology I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Ralf P
authors: RP
authors: Brandes
authors: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.
authors: Bernhard
authors: B
authors: Brüne
authors: Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany. Electronic address: vonknethen@biochem.uni-frankfurt.de.
authors: Andreas
authors: A
authors: von Knethen
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D001853:Bone Marrow / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003553:Cystine / Q000378:metabolism*; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D051267:NF-E2-Related Factor 2 / Q000502:physiology*; D010047:Ovalbumin / Q000276:immunology / Q000378:metabolism; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D000975:Antioxidants; D015395:Histocompatibility Antigens Class I; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D011948:Receptors, Antigen, T-Cell; D003553:Cystine; D010047:Ovalbumin; D005978:Glutathione
keywords: BMDMΦ; CD8(+) T cells; Cysteine; Cytotoxicity; Free radicals; Glutathione; Nrf2; T cell tolerance; xCT; γ-GCS