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Updated: Jun 12, 2026
Culture of myeloid dendritic cells from bone marrow precursors
Published on: July 25, 2008
Single-Cell Analysis Reveals a Subset of High IL-12p40-Secreting Dendritic Cells within Mouse Bone Marrow-Derived
Kate Bridges1, Gabriela A Pizzurro1, Mihir Khunte1
1Department of Biomedical Engineering, Yale University, New Haven, CT.
Abstract:
Macrophages and dendritic cells (DCs), although ontogenetically distinct, have overlapping functions and exhibit substantial cell-to-cell heterogeneity that can complicate their identification and obscure innate immune function. In this study, we report that M-CSF-differentiated murine bone marrow-derived macrophages (BMDMs) exhibit extreme heterogeneity in the production of IL-12, a key proinflammatory cytokine linking innate and adaptive immunity. A microwell secretion assay revealed that a small fraction of BMDMs stimulated with LPS secrete most IL-12p40, and we confirmed that this is due to extremely high expression of Il12b, the gene encoding IL-12p40, in a subset of cells. Using an Il12b-YFP reporter mouse, we isolated cells with high LPS-induced Il12b expression and found that this subset was enriched for genes associated with the DC lineage. Single-cell RNA sequencing data confirmed a DC-like subset that differentiates within BMDM cultures that is transcriptionally distinct but could not be isolated by surface marker expression. Although not readily apparent in the resting state, upon LPS stimulation, this subset exhibited a typical DC-associated activation program that is distinct from LPS-induced stochastic BMDM cell-to-cell heterogeneity. Overall, our findings underscore the difficulty in distinguishing macrophages and DCs even in widely used in vitro murine BMDM cultures and could affect the interpretation of some studies that use BMDMs to explore acute inflammatory responses.
Insights
Murine bone marrow-derived macrophages (BMDMs) show significant heterogeneity in IL-12 production, with a small subset exhibiting dendritic cell (DC)-like properties. This challenges the clear distinction between macrophages and DCs in in vitro studies.
Area of Science:
- Immunology
- Cell Biology
- Innate Immunity
Background:
- Macrophages and dendritic cells (DCs) have overlapping functions and heterogeneity, complicating identification and innate immune studies.
- Interleukin-12 (IL-12) is a key proinflammatory cytokine linking innate and adaptive immunity.
- Murine bone marrow-derived macrophages (BMDMs) are widely used in vitro models for studying immune responses.
Purpose of the Study:
- To investigate the heterogeneity of IL-12 production in M-CSF-differentiated BMDMs.
- To identify the cellular and molecular basis for IL-12 production variability within BMDM cultures.
- To determine if a distinct subset within BMDMs exhibits dendritic cell (DC)-like characteristics.
Main Methods:
- Microwell secretion assay to measure IL-12p40 production in LPS-stimulated BMDMs.
- Il12b-YFP reporter mouse to isolate cells with high Il12b expression.
- Single-cell RNA sequencing (scRNA-seq) to analyze transcriptional profiles of BMDM subsets.
- Analysis of gene expression associated with DC lineage and activation programs.
Main Results:
- BMDMs exhibit extreme heterogeneity in IL-12p40 secretion upon LPS stimulation.
- A small subset of BMDMs demonstrates high Il12b gene expression, correlating with DC-associated gene signatures.
- scRNA-seq identified a transcriptionally distinct, DC-like subset within BMDM cultures, not identifiable by surface markers.
- This DC-like subset displayed a distinct activation program upon LPS stimulation, separate from stochastic BMDM heterogeneity.
Conclusions:
- Distinguishing between macrophages and DCs in in vitro BMDM cultures is challenging due to inherent heterogeneity.
- A subset of BMDMs can acquire DC-like properties and activation programs, impacting the interpretation of inflammatory response studies.
- These findings highlight the need for careful consideration of cellular heterogeneity in interpreting in vitro immunology experiments.
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