Background/Purpose: Monocytes are essential to innate immunity but also propagate the inflammatory response in autoimmune arthritis and crystal arthropathies.  Development of inflammatory monocytes (Ly6C^hi CCR2⁺) and residential monocytes (Ly6C^lo CX₃CR1⁺) from murine bone marrow precursor cells is an enigmatic process that involves numerous transcription factors with influence by growth factors and cytokines. Elucidation of these critical pro-inflammatory pathways is limited by the paucity of precursor cells and the need for extensive ex-vivo manipulation and cell sorting.

Methods: To develop an informative myeloid cell line, we infected bone marrow from CCR2^RFP/+ CX₃CR1^GFP/+ dual-reporter mice with a murine stem cell virus encoding Hoxb8 under the control of estrogen receptor. These cells remain immortalized at the myeloid precursor stage until induced by estrogen withdrawal to initiate synchronous myeloid differentiation. To identify pathways important to monocyte development, small molecule inhibitors, cytokines and growth factors were added to cell culture at specific timepoints, and key findings were confirmed in live mice.

Results: Upon cessation of Hoxb8 expression after estrogen withdrawal, our novel dual-reporter cell line spontaneously differentiated into Ly6C^hi CCR2⁺ inflammatory monocytes, followed by maturation to Ly6C^lo CX₃CR1⁺ residential monocytes.  Using this cell line as a screening tool, we identified an essential role of phosphoinositide-3 kinase (PI3K) and mechanistic target of rapamycin (mTOR) in spontaneous monocyte development through a small molecule library screen.  These findings were confirmed in vivo as mice with conditional deletion of mTOR complex 1, but not mice lacking mTOR complex 2, failed to develop Ly6C^hi monocytes.  While macrophage colony-stimulating factor (M-CSF) was not required for spontaneous production of monocytes, the addition of exogenous M-CSF induced Ly6C^hi monocyte development via a PI3K/mTOR-independent pathway.

Conclusion: We describe a novel dual-reporter myeloid progenitor cell line that recapitulates the monocyte development continuum. Studies using these cells enabled identification of a previously unrecognized role of mTORC1 in normal development of inflammatory monocytes, as well as a potential “rescue” pathway of mTORC1-independent differentiation.  Our work highlights the utility of this technology in studying myeloid differentiation and provides evidence for the existence of two distinct pathways of monocyte production that may be differentially employed in physiologic and inflammatory conditions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/distinct-pathways-of-ly6chi-monocyte-development-identified-by-a-novel-dual-reporter-murine-myeloid-progenitor-cell-line/