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Essential Role of Mtorc1 in Self-Renewal of Murine Alveolar Macrophages

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Essential Role of Mtorc1 in Self-Renewal of Murine Alveolar Macrophages The Journal of Immunology Essential Role of mTORC1 in Self-Renewal of Murine Alveolar Macrophages Wenhai Deng,*,† Jialong Yang,† Xingguang Lin,* Jinwook Shin,† Jimin Gao,* and Xiao-Ping Zhong†,‡,x Alveolar macrophages (AMf) have the capacity of local self-renewal through adult life; however, mechanisms that regulate AMf self-renewal remain poorly understood. We found that myeloid-specific deletion of Raptor, an essential component of the mammalian/mechanistic target of rapamycin complex (mTORC)1, resulted in a marked decrease of this population of cells accompanying altered phenotypic features and impaired phagocytosis activity. We demonstrated further that Raptor/mTORC1 deficiency did not affect AMf development, but compromised its proliferative activity at cell cycle entry in the steady-state as well as in the context of repopulation in irradiation chimeras. Mechanically, mTORC1 confers AMf optimal responsiveness to GM- CSF–induced proliferation. Thus, our results demonstrate an essential role of mTORC1 for AMf homeostasis by regulating proliferative renewal. The Journal of Immunology, 2017, 198: 492–504. lveolar macrophages (AMf) are the most abundant first week after birth. These differentiated AMf persist through immune cells residing in terminal airways, where they adulthood via proliferative self-renewal independent of circulating A play important functions in lung development, integrity, monocytes (11). However, under certain conditions, such as bone surfactant metabolism, and host defense responses, rendering marrow transplantation after lethal irradiation, AMf can be them prominent targets for therapeutic intervention (1, 2). The replenished from bone marrow–derived monocytes (8, 12, 13), traditional view that AMf belong to the mononuclear phagocyte which serves as an emergency pathway of AMf ontogeny. During system (3) with bone marrow–derived monocytes as develop- AMf development, they undergo profound changes of the surface mental intermediates has been recently challenged. Accumulating profile, which are characterized by increased expression of evidence has recently indicated that many tissue-resident macro- CD11c, Siglec-F, F4/80, and CD64, and concomitantly downreg- phages, including AMf, are derived from embryonic precursors ulation of CD11b (11, 13). Local environmental signals, such as and are self-maintained with minimal contribution from circulat- GM-CSF, instruct AMf via PPAR-g to acquire such signature ing bone marrow–derived precursors in steady-states (4–10). Fetal phenotypes and functions (13–15). Moreover, GM-CSF is also monocytes, as AMf precursors, seed into the lung prior to birth, required for AMf maintenance in promoting proliferation (8, 16– expand massively, and then develop into mature AMf during the 19). Although emerging evidence highlights proliferative self- renewal as the main mechanism for AMf maintenance in both steady-state and under stress conditions, mechanisms that link *School of Laboratory Medicine, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China; †Division of Allergy and Immunology, Department of Pediatrics, mitogenic stimuli, such as GM-CSF, to proliferative renewal Duke University Medical Center, Durham, NC 27710; ‡Department of Immunology, programming remain largely unknown. x Duke University Medical Center, Durham, NC 27710; and Hematologic Malignan- Proliferating animal cells must tightly coordinate cell cycle cies and Cellular Therapies Program, Duke Cancer Institute, Duke University Med- ical Center, Durham, NC 27710 progression with cell growth and proliferation-associated bioen- ORCIDs: 0000-0003-3476-7595 (J.S.); 0000-0002-3911-043X (J.G.); 0000-0002- ergetic demand. Mechanistic target of rapamycin (mTOR), a highly 4619-8783 (X.-P.Z.). conserved serine-threonine kinase, serves a key sensor for meta- Received for publication August 18, 2015. Accepted for publication November 2, bolic cues to regulate cell growth and proliferation (20, 21). mTOR 2016. forms at least two known distinct complexes, mTOR complex This work was supported by National Institute of Allergy and Infectious Diseases/ (mTORC)1 and mTORC2. mTORC1 contains mTOR, Deptor, National Institutes of Health Grants R01 AI079088 and R01 AI101206. mLST8, PRAS40, and the adapter protein Raptor, and it is sen- W.D. designed and performed experiments, analyzed data, and wrote the manuscript; sitive to the immunosuppressant rapamycin. mTORC1 acts J.Y., X.L., and J.S. performed experiments; J.G. participated in data analysis; X.-P.Z. designed experiments, participated in data analysis, and wrote the manuscript. downstream of the PI3K–Akt–Tsc1/2 pathway to phosphorylate Address correspondence and reprint requests to Xiao-Ping Zhong, Division of Al- translational regulators, the S6 ribosomal kinase, and the trans- lergy and Immunology, Department of Pediatrics, Duke University Medical Center, lation initiation factor 4E binding protein 1 (4E-BP1) (22). Sub- Box 2644, Durham, NC 27710 or Jimin Gao at the current address: School of sequently, S6 ribosomal kinase phosphorylates the ribosomal Laboratory Medicine, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China. E-mail addresses: [email protected] (X.-P.Z.) or jimingao@yahoo. protein S6 to promote ribosomogenesis. Furthermore, activation of com (J.G.) mTORC1 promotes the downstream anabolic processes, such as The online version of this article contains supplemental material. glycolysis, by activating the transcriptional factors Hif1a and Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; AMf, alveolar c-Myc, as well as de novo lipid biosynthesis via upregulating macrophage; 4E-BP1, 4E binding protein 1; gMFI, geometric mean fluorescence SREBPs, while suppressing catabolic processes such as autophagy intensity; IMf, interstitial macrophage; LPf, large peritoneal macrophage; mTOR, mammalian/mechanistic target of rapamycin; mTORC, mammalian/mechanistic tar- (20, 21, 23, 24). As such, essential roles of mTORC1 and its tight get of rapamycin complex; 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) regulation by Tsc1 have been demonstrated to regulate both innate amino)-2-deoxyglucose; 4-OHT, 4-hydroxy-tamoxifen; qPCR, quantitative PCR; and adaptive immunity (25–40). Whereas inhibition of mTORC1 RPf, red pulp macrophage; WT, wild-type. can reduce proinflammatory cytokine production and M1 polari- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 zation by macrophages, constitutive mTORC1 activation due to www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501845 Downloaded by guest on October 1, 2021 The Journal of Immunology 493 Tsc1 deletion leads to enhanced proinflammatory responses and viability kit or 7-aminoactinomycin D (7-AAD; Invitrogen) was used for macrophage M1 polarization but resistance to IL-4–induced M2 the exclusion of dead cells. For assessment of apoptosis, annexin V and polarization and endotoxin tolerance (41–44). Despite extensive FITC-VAD-FMK (Promega) staining kits were applied. For detection of intracellular cytokine or phosphorylated signaling progress in our understanding of the role of mTORC1 in macrophage proteins, freshly isolated cells or stimulated cells were fixed using BD function, the importance of mTORC1 signaling in the development or Cytofix/Cytoperm buffer according to the manufacturer’s instructions. maintenance of macrophages is largely unclear. In this study, we Following permeabilization with prechilled pure methanol (220˚C) for at demonstrated an essential role of mTORC1 in AMf homeostasis, at least 30 min on ice, intracellular staining was performed with Abs, including anti-IL-12 (p40)–PE (C15.6; BioLegend), anti-pS6 (Ser235/236)–Alexa Fluor least in part by promoting their proliferative self-renewal via ensuring 647 (D57.2.2E; Cell Signaling Technology), and anti–p-4E-BP1 (Thr37/46; optimal responsiveness to GM-CSF–induced cell cycle entry. 236B4; Cell Signaling Technology). Anti–p-4E-BP1 was detected with anti-rabbit IgG–Alexa Fluor 594 (Invitrogen). Isotype-matched control Abs were used as a negative control. Materials and Methods We noted that Raptor deficiency AMf exhibited elevated auto- Mice fluorescence compared with wild-type (WT) controls (Fig. 2A, Supplemental fl/fl cre/+ Zsgreen/+ Fig. 2A). As a source of noise, autofluorescence may decrease sensitivity to Rptor , LyzM , R26 (containing the robust bright reporter distinguish the levels of individual Ag markers (45). To unequivocally de- gene Zsgreen following the STOP cassette driven by the endogenous termine the surface phenotypes of AMf, we adapted fluorescence minus one Rosa26 promoter) mice were purchased from The Jackson Laboratory. staining sets in which a channel of interest was stained with isotype Abs to Rptor fl/fl mice were crossed with LyzMcre/+ mice or R26CreERT2/+ mice to fl/fl cre/+ DMyel fl/fl establish autofluorescence control, and the expression of each marker was generate Rptor LyzM (called Rptor in this study) or Rptor quantified by dividing the geometric mean fluorescence intensity (gMFI) of R26CreERT2/+ mice, respectively. To monitor the activity of Cre recombi- cre/+ DMyel Zsgreen/+ the molecule of interest of the stained cells by the gMFI of the fluorescence nase, LyzM and Rptor mice were further mated with R26 minus one control. Data were acquired on BD FACSCanto II and analyzed mice. All mice were in the C57BL/6 genetic background. Experiments with FlowJo software (Tree Star). were carried on 6- to 9-wk-old mice unless otherwise indicated. All pro- cedures were in compliance with protocols
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