The metabolic regulator mTORC1 controls terminal myeloid differentiation

Abstract

Monocytes are derived from hematopoietic stem cells through a series of intermediate progenitor stages, but the factors that regulate this process are incompletely defined. Using a Ccr2/Cx3cr1 dual-reporter system to model murine monocyte ontogeny, we conducted a small-molecule screen that identified an essential role of mechanistic target of rapamycin complex 1 (mTORC1) in the development of monocytes and other myeloid cells. Confirmatory studies using mice with inducible deletion of the mTORC1 component Raptor demonstrated absence of mature circulating monocytes, as well as disruption in neutrophil and dendritic cell development, reflecting arrest of terminal differentiation at the granulocyte-monocyte progenitor stage. Conversely, excess activation of mTORC1 through deletion of the mTORC1 inhibitor tuberous sclerosis complex 2 promoted spontaneous myeloid cell development and maturation. Inhibitor studies and stage-specific expression profiling identified failure to down-regulate the transcription factor Myc by the mTORC1 target ribosomal S6 kinase 1 (S6K1) as the mechanistic basis for disrupted myelopoiesis. Together, these findings define the mTORC1-S6K1-Myc pathway as a key checkpoint in terminal myeloid development.

High-dimensional analysis reveals a pathogenic role of inflammatory monocytes in experimental diffuse alveolar hemorrhage

“…Flow cytometry. The protocol for flow cytometry was previously described (61), and fluorophore-conjugated antibodies used are listed in Supplemental Table 1. In brief, cells were incubated with anti-mouse CD16/32, then stained with an optimized amount of primary antibody or the appropriate isotype control for 15 minutes at room temperature before washing and resuspending in PBS supplemented with 0.1% BSA.…”

“…qPCR. The protocol for qPCR was described previously (61). Total RNA was extracted from peritoneal exudate cells using RNeasy Mini Kit (Qiagen), and cDNA was synthesized using the Superscript III First-Strand Synthesis Kit (Thermo Fisher Scientific).…”

“…RNA-Seq data were deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (accession number GSE133083). The protocol for bulk RNA-Seq was described previously (61). In brief, RNA from peritoneal cells (1 × 10 5 ) was isolated using Qiagen RNeasy Micro Kit.…”

“…Smart-Seq2 libraries were prepared by the Broad Technology Labs (BTL) and sequenced by the Broad Genomics Platform. Transcripts were quantified by the BTL computational pipeline (61). GSEA was performed using software v3.0 (Broad Institute) with the Molecular Signatures Database hallmark gene set collection (62).…”

Tumor Microenvironment Modulates Immunological Outcomes of Myeloid Cells with mTORC1 Disruption

“…These data suggest that mTORC1 signaling in myeloid cells is important for the monocytes' differentiation into TAM in the s.c tumors. The CSF1/CSF1 receptor (CSF1R) axis plays essential roles in TAM differentiation, and mTORC1 signaling has been shown to be critical in M-CSF-mediated myelopoiesis (26). Thus, we hypothesized that decreased TAM frequency in mTORC1 cKO s.c. tumors may be the result of downregulation of CSF1R in Raptor-deficient macrophages.…”