title: Analysis of single-cell cytokine secretion reveals a role for paracrine signaling in coordinating macrophage responses to TLR4 stimulation.
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.
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Xue
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
authors.forename: Yao
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. Institute for System Dynamics, University of Stuttgart, 70569 Stuttgart, Germany.
authors.forename: Markus R
authors.initials: MR
authors.lastname: Eisele
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
authors.forename: Endah S
authors.initials: ES
authors.lastname: Sulistijo
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.
authors.forename: Nafeesa
authors.initials: N
authors.lastname: Khan
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. rong.fan@yale.edu kathryn.miller-jensen@yale.edu.
authors.forename: Rong
authors.initials: R
authors.lastname: Fan
authors.affiliation: 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.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Miller-Jensen
mesh_terms: D016207:Cytokines / Q000276:immunology*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000276:immunology*; D019899:Paracrine Communication / Q000187:drug effects* / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000276:immunology*; D020298:U937 Cells
chemical_list: D016207:Cytokines; D008070:Lipopolysaccharides; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4