Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response

Cell Rep. 2021 Sep 21;36(12):109728. doi: 10.1016/j.celrep.2021.109728.


Following Toll-like receptor 4 (TLR4) stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine interleukin-10 (IL-10) limits the inflammatory response. However, extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterize the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enables evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data reveals three tiers of TLR4-induced proinflammatory activation based on levels of cytokine secretion. Surprisingly, while IL-10 inhibits TLR4-induced activation in the highest tier, it also contributes to the TLR4-induced activation threshold by regulating which cells transition from non-secreting to secreting states. This role for IL-10 in restraining TLR4 inflammatory activation is largely mediated by intermediate interferon (IFN)-β signaling, while TNF likely mediates response resolution by IL-10. Thus, cell-to-cell variability in cytokine regulatory motifs provides a means to tailor the TLR4-induced inflammatory response.

Keywords: IL-10; TLR4 signaling; macrophage; single-cell measurements.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antibodies / immunology
  • Antibodies / pharmacology
  • Chemokine CCL5 / metabolism
  • Female
  • Interferon-beta / metabolism
  • Interleukin-10 / genetics
  • Interleukin-10 / metabolism*
  • Interleukin-10 / pharmacology
  • Lipopolysaccharides / pharmacology
  • Macrophages / cytology
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Receptors, Interleukin-10 / immunology
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology
  • Signal Transduction / drug effects
  • Single-Cell Analysis
  • Toll-Like Receptor 4 / metabolism*
  • Tumor Necrosis Factor-alpha / metabolism


  • Antibodies
  • Chemokine CCL5
  • Lipopolysaccharides
  • Receptors, Interleukin-10
  • Recombinant Proteins
  • Toll-Like Receptor 4
  • Tumor Necrosis Factor-alpha
  • Interleukin-10
  • Interferon-beta