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

Following TLR4 stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine 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 characterized the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enabled evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data revealed three distinct 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 IFN-{beta} signaling, while TNF-a 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.