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

Abstract

Cellular responses are mediated by heterogeneous intermediate signals that are secreted and sensed by the same cells. Cell-to-cell communication through these intermediate signals likely affects the collective response of cells within a population. We combined multiplexed, microwell-based measurements of cytokine secretion by single cells with data from the analysis of cell populations to determine the role of paracrine signaling in shaping the profile of inflammatory cytokines secreted by macrophages in response to the stimulation of Toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS). Loss of paracrine signaling as a result of cell isolation substantially reduced the secretion of a subset of LPS-stimulated cytokines, including interleukin-6 (IL-6) and IL-10, by macrophage-like U937 cells and human monocyte-derived macrophages (MDMs). 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 identified as the most influential cytokine in the GGM network, and our data suggest that paracrine signaling from a small subpopulation of “high-secreting” cells, which generated most of the TNF-α produced, was necessary but not sufficient to achieve high secretion of IL-6 and IL-10 in the cell population. Decreased IL-10 secretion in 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 and reliable innate immune response in spite of underlying cell-to-cell heterogeneity.

Systematic Investigation of Multi-TLR Sensing Identifies Regulators of Sustained Gene Activation in Macrophages

“…This may suggest an important role for autocrine/paracrine type I interferon and other cytokine responses which could feedback on the TLR-activated macrophage to modulate the non-additive immune effector responses promoted by combined TLR activation (Xue et al, 2015). Such mediators likely also perform a key function in innate/adaptive immune cell communication through leukocyte recruitment and activation, as demonstrated by the selective functional annotation of the synergized gene clusters derived from ingenuity pathway analysis (IPA) (Figure 3C).…”

What Scales the T Cell Response?

“…Paracrine messengers secreted from cells that produce them in larger quantities can either trigger secretion of a secondary messenger from nonproducers and/or trigger responses in all the cells in the vicinity because of diffusive spread [49,50]. Thus, a paracrine messenger can be a shared messenger for a collective, coherent response.…”

Redefining Signaling Pathways with an Expanding Single-Cell Toolbox

“…We found that the total secretion of IL-6 and IL-10 was lower compared to conventional cultures, demonstrating that loss of paracrine signals attenuates inflammatory signaling (Fig. 2E; [46]). Heterogeneity of TNF secretion was especially high, with a few cells accounting for a majority of TNF secretion in the population.…”