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Chronic Innate Immune Activation of TBK1 Suppresses Mtorc1 Activity and Dysregulates Cellular Metabolism

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Chronic Innate Immune Activation of TBK1 Suppresses Mtorc1 Activity and Dysregulates Cellular Metabolism Chronic innate immune activation of TBK1 suppresses mTORC1 activity and dysregulates cellular metabolism Maroof Hasana,1,2, Vijay K. Gonuguntaa,1, Nicole Dobbsa, Aktar Alib, Guillermo Palchikc, Maria A. Calvarusod, Ralph J. DeBerardinisd,e,f, and Nan Yana,g,3 aDepartment of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390; bDepartment of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; cDepartment of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390; dChildren’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390; eDepartment of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390; fMcDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX 75390; and gDepartment of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390 Edited by Michael Karin, University of California, San Diego School of Medicine, La Jolla, CA, and approved December 9, 2016 (received for review July 7, 2016) Three-prime repair exonuclease 1 knockout (Trex1−/−) mice suffer Here, we show that mTORC1 activity is reduced in multiple −/− from systemic inflammation caused largelybychronic activation of Trex1 mouse tissues and TREX1 mutant human cells. We also −/− the cyclic GMP-AMP synthase–stimulator of interferon genes–TANK- show that Trex1 mice exhibit many metabolic-dysregulation binding kinase–interferon regulatory factor 3 (cGAS–STING–TBK1– phenotypes consistent with chronically reduced mTORC1 activity, IRF3) signaling pathway. We showed previously that Trex1-deficient including reduced cellular mitochondrial respiration and increased cells have reduced mammalian target of rapamycin complex 1 in vivo energy expenditure and fat metabolism, leading to reduced (mTORC1) activity, although the underlying mechanism is unclear. adiposity and survival. We genetically separated inflammation and −/− metabolic defects and demonstrate that metabolic dysregulation in Here, we performed detailed metabolic analysis in Trex1 mice −/− and cells that revealed both cellular and systemic metabolic defects, Trex1 mice is caused by innate immune activation of TBK1, but not the downstream IRF3 signaling and inflammation, and that TBK1 including reduced mitochondrial respiration and increased glycolysis, −/− energy expenditure, and fat metabolism. We also genetically sepa- binds to the mTORC1 complex and inhibits its activity in Trex1 cells. Our data thus establish TBK1–mTORC1 as an important reg- rated the inflammatory and metabolic phenotypes by showing that ulatory axis between cell-intrinsic immune signaling and metabolism. Sting deficiency rescued both inflammatory and metabolic pheno- types, whereas Irf3 deficiency only rescued inflammation on the −/− Results Trex1 background, and many metabolic defects persist in −/− −/− −/− Reduced mTORC1 Activity and Cellular Metabolism in Trex1 Cells Trex1 Irf3 cells and mice. We also showed that Leptin deficiency −/− − − and Tissues. Recently, we showed that Trex1 cells have reduced (ob/ob) increased lipogenesis and prolonged survival of Trex1 / mTORC1 activity, but the underlying mechanism is unclear (1). mice without dampening inflammation. Mechanistically, we identi- −/− − − Trex1 mice also display reduced body size and weight that / − − fied TBK1 as a key regulator of mTORC1 activity in Trex1 cells. resembles reduced mTORC1 function as in S6K / or Raptor Together, our data demonstrate that chronic innate immune activa- conditional knockout mice (8, 9) (Fig. 1A). To extend our earlier tion of TBK1 suppresses mTORC1 activity, leading to dysregulated in vitro findings, we first evaluated the mTORC1 activity in tis- − − cellular metabolism. sues of Trex1 / mice. We observed clear reduction in mTORC1 activity (measured by S6P and 4EBP phosphorylation) in the − − TREX1 | TBK1 | mTORC1 | innate immunity | metabolism liver, skeletal muscles, and kidney of Trex1 / mice (Fig. 1B and ammals have evolved complex and integrated systems of Significance Mimmunity and metabolism to maintain and defend internal and environmental threats. Increasing numbers of immune regulators have Patients with chronic autoimmune and autoinflammatory dis- been found to play critical roles in metabolism. Studies from recent eases often also present metabolic phenotypes. The molecular years have established an integrated view on the shared architecture connection between inflammation and metabolism is incom- between adaptive immunity and metabolism, including how they cor- pletely understood. We describe a mouse model, three-prime respond to stimulus in disease settings. However, cell-intrinsic innate repair exonuclease 1 knockout, that presents both chronic sys- immune regulation of metabolic pathways remains poorly understood. temic inflammation and metabolic dysregulation. We genetically This is in part due to the fact that overwhelming systemic inflammation separated the inflammation and metabolic phenotypes and in chronic diseases often masks direct underlying molecular causes. biochemically identified TANK-binding kinase 1 (TBK1) as a key We have previously characterized an autoimmune/autoinflammatory regulator of mammalian target of rapamycin complex 1, a disease mouse model, three-prime repair exonuclease 1 knockout master regulator of metabolism. Chronically activated TBK1 and −/− (Trex1 )(1).TREX1,alsoknownasDNaseIII,isanexonuclease interferon signaling are associated with many autoimmune dis- that localizes to the endoplasmic reticulum (ER) to prevent aberrant eases, including systemic lupus erythematosus. Our study pro- accumulation of self-DNA or glycans that would trigger immune vides a mechanism by which the innate immune signaling activation (2, 3). Mutations of the TREX1 gene have been associated pathways regulate cellular metabolism. with several autoinflammatory and autoimmune diseases, including Aicardi–Goutières syndrome and systemic lupus erythematosus Author contributions: M.H., V.K.G., and N.Y. designed research; M.H., V.K.G., N.D., A.A., − − (SLE) (4). Trex1 / mice suffer from systemic inflammation caused G.P., and M.A.C. performed research; A.A. and R.J.D. contributed new reagents/analytic largely by chronic activation of the cytosolic DNA sensing pathway tools; M.H., V.K.G., N.D., G.P., M.A.C., and N.Y. analyzed data; and M.H., V.K.G., and N.Y. through the cyclic GMP-AMP synthase–stimulator of interferon wrote the paper. genes–TANK-binding kinase–interferon regulatory factor 3 (cGAS– The authors declare no conflict of interest. STING–TBK1–IRF3) cascade (5–7). We showed previously that This article is a PNAS Direct Submission. TREX1 also regulates lysosomal biogenesis through the mTORC1– 1M.H. and V.K.G. contributed equally to this work. −/− TFEB pathway and that Trex1 mouse embryonic fibroblasts 2Present address: Novartis Institutes of BioMedical Research, Basel, Switzerland. (MEFs) exhibit drastically reduced mTORC1 activity compared with 3To whom correspondence should be addressed. Email: [email protected]. WT cells (1). The molecular mechanism leading to suppression of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mTORC1 activity remains unknown. 1073/pnas.1611113114/-/DCSupplemental. 746–751 | PNAS | January 24, 2017 | vol. 114 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1611113114 Downloaded by guest on September 25, 2021 ABCD E F G HI − − − − Fig. 1. Reduced mTORC1 activity and defective mitochondrial respiration in Trex1 / cells and tissues. (A) Representative images of WT and Trex1 / mice at the − − age of 6–8 wk. Body weight was measured at 8 wk (n = 10). (B) Immunoblot analysis of phosphorylated and total S6P and 4E-BP1 in WT and Trex1 / mouse tissues. (C) Immunoblot analysis of phosphorylated and total mTOR and S6P in healthy control (HC) and TREX1-D18N and TREX1-D200H mutant patient lym- phoblasts. (D) Immunoblot analysis of phosphorylated and total S6P in Trex1−/− MEFs reconstituted with GFP (control) or mouse TREX1. (E–G) Real-time changes in theOCRandECARofWTorTrex1−/− BMDMs (E and F)orMEFs(G). OCR was assessed during subsequent sequential treatment with oligomycin (inhibitor of ATP synthase), FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), and rotenone (inhibitors of the electron-transport chain). ECAR was assessed during sequential treatment with 25 mM glucose and 2-deoxyglucose (2-DG). (H) Schematic representation of metabolism of [U-13C]glucose for experiments in G.(I) Mass isotopomer analysis of citrate, fumarate, and malate in cells cultured with [U-13C]glucose and unlabeled glutamine. Data are presented as the means ± SEM of quadruplets of two to three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t test). Fig. S1 A and B). Interestingly, AKT phosphorylation is un- measured by extracellular acidification rate (ECAR) (Fig. 1F). The − − − − changed in Trex1 / tissues where mTORC1 activity is clearly mitochondrial-respiration defect in Trex1 / MEFs was completely reduced, indicating that mTORC1 is regulated independent of restored by reexpressing WT TREX1 (Fig. 1G). To confirm the AKT in these tissues (Fig. S1B). We observed some reduction mitochondrial defect and pinpoint the specific step(s) of cellular − − − − of 4E–BP phosphorylation in Trex1 / bone marrow-derived metabolism that might be affected by Trex1 / , we performed 13C dendritic cells (BMDCs), and in adipose tissue,
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