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Sci Signal. 2015 Jun 16;8(381):ra59. doi: 10.1126/scisignal.aaa2155.

Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation.

Author information

1
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
2
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. Institute for System Dynamics, University of Stuttgart, 70569 Stuttgart, Germany.
3
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. rong.fan@yale.edu kathryn.miller-jensen@yale.edu.
4
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA. rong.fan@yale.edu kathryn.miller-jensen@yale.edu.

Abstract

Macrophages not only produce multiple cytokines but also respond to multiple cytokines, which likely shapes the ultimate response of the population. To determine the role of paracrine signaling in shaping the profile of inflammatory cytokines secreted by macrophages in response to stimulation of Toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS), we combined multiplexed, microwell-based measurements of cytokine secretion by single cells with analysis of cytokine secretion by cell populations. Loss of paracrine signaling as a result of cell isolation reduced the secretion by macrophage-like U937 cells and human monocyte-derived macrophages (MDMs) of a subset of LPS-stimulated cytokines, including interleukin-6 (IL-6) and IL-10. Graphical Gaussian modeling (GGM) of the single-cell data defined a regulatory network of paracrine signals, which was validated experimentally in the population through antibody-mediated neutralization of individual cytokines. Tumor necrosis factor-α (TNF-α) was the most influential cytokine in the GGM network. Paracrine signaling by TNF-α secreted from a small subpopulation of "high-secreting" cells was necessary, but not sufficient, for the secretion of large amounts of IL-6 and IL-10 by the cell population. Decreased relative IL-10 secretion by isolated MDMs was linked to increased TNF-α secretion, suggesting that inhibition of the inflammatory response also depends on paracrine signaling. Our results reveal a previously uncharacterized role for cell-to-cell communication within a population in coordinating a rapid innate immune response despite underlying cell-to-cell heterogeneity.

PMID:
26082435
PMCID:
PMC5735825
DOI:
10.1126/scisignal.aaa2155
[Indexed for MEDLINE]
Free PMC Article

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