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

Abstract

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.

Figure 1.. IL-10 suppresses TLR4-induced proinflammatory activation in a dose-dependent manner

(A) Schematic of paracrine signaling cascades activated by TLR4 stimulation. (B) Time course of TNF secretion following stimulation of BMDMs at the indicated LPS dose. TNF secretion was measured by ELISA and is presented as mean ± standard error of the mean (SEM) of 3 biological replicates. (C) Area under the curve (AUC) calculated from TNF secretion over time for each dose in (B). (D) Dose response of TNF and IL-10 secretion following LPS stimulation of BMDMs for 8 h (left) and 24 h (right). Protein secretion was measured by ELISA and is presented as mean ± SEM of 3 biological replicates. Hill slopes calculated from 4-parameter logistic curves fitted to the dose-response data.

Figure 2.. Single-cell secretion analysis reveals tiered activation of the TLR4 secretion program

(A) Schematic of workflow for single-cell secretion profiling. BMDMs were seeded onto the microwell chip and then stimulated with 0, 10, 100, or 1,000 ng/mL LPS for 8 h. High-dimensional secretion data were visualized by PHATE. (B) Violin plots of single-cell secretion from BMDMs stimulated with LPS in the microwell assay. Black bar indicates fluorescent threshold of detection. Data are pooled from 3 independent biological replicates. (C) Four-parameter logistic curves were interpolated from the mean percentage of cells secreting TNF and IL-10 above threshold in response to the indicated LPS dose. Data presented as mean ± 95% confidence intervals (CIs) calculated by bootstrapping. (D and E) 2D PHATE visualization of 10-dimensional single-cell secretion from BMDMs stimulated with LPS as in (A) (four conditions). Data include activated macrophages (i.e., secreting at least 1 measured cytokine above threshold) colored by (D) relative intensity of the indicated cytokine (non-zero cells brought to front for visualization) or (E) the KDE for cells at the indicated LPS dose (other doses shown in gray). (F)Scatterplots of the number of proteins co-secreted in the microwell device versus PHATE 1 coordinate. Spearman correlation = 0.85 (p value < 0.0001). (G)KDE for individual BMDMs along the PHATE 1 axis calculated from data (E).

Figure 3.. IL-10 negative feedback modulates heterogeneity in the TLR4 secretion program

(A) 2D PHATE visualization of 9-dimensional single-cell secretion data for nine conditions: BMDMs stimulated for 8 h with LPS alone (10, 100, or 1,000 ng/mL), co-stimulated with IL-10R Ab (30 μg/mL) at each dose, or co-stimulated with recombinant IL-10 (10 ng/mL) at each dose. Data are colored by KDE for the 100 ng/mL LPS conditions (other cells grayed out for visualization). (B) KDE for individual BMDMs along the PHATE 1 axis calculated from 100 ng/mL LPS stimulation shown in (A) and from 1,000 and 10 ng/mL LPS stimulations (Figure S3B). Arrows indicate inter-mediate and high modes of the trimodal PHATE 1 KDE distribution. The p values were calculated using the Kolmogorov-Smirnov test to determine whether two samples come from the same distribution.

Figure 4.. Low versus high levels of IL-10 negative feedback differentially modulate TNF activation

(A) Probability histograms for TNF secretion after 8 h of the indicated stimulation in the microwell device. Non-responding cells were set to 1 (“OFF”). Inset graphs show TNF+ subpopulation. (B) Bargraphs of average percentage of cells secreting the protein above threshold in response to the indicated stimulation cues after 8 h in the microwell assay (top) or mean secretion level from the responding subpopulation of cells (only cells secreting indicated cytokine/chemokine above detection threshold in microwell device) (bottom). Data presented as mean ± 95% CIs calculated by bootstrapping. Significance determined by non-overlap of CIs. (C) Conditional density re-scaled visualization (DREVI) plots showing the relationship between IL-10 and TNF. Data shown are pooled to include all stimulation doses (0, 10, 100, and 1,000 ng/mL LPS with or without IL-10R Ab at 30 μg/mL) where the 2 indicated cytokines were co-secreted during the 8-h stimulation (LPS alone: n = 516, +IL-10R Ab: n = 212). Arrows indicate areas of low and high IL-10 secretion most affected by IL-10 negative feedback. (D) Edge response functions for DREVI plots shown in (C) were fit according to the conditional mean at the region of highest conditional density.

Figure 5.. IFN-β largely mediates IL-10 regulation of the threshold for TLR4 activation, while TNF positive feedback activates the resolving role of IL-10

(A and B) 2D PHATE projection of 9-dimensional single-cell secretion data from BMDMs stimulated with 100 ng/mL LPS alone or co-stimulated with 30μg/mL IL-10R Ab, 5 μg/mL IFNAR Ab, or 5 μmg/mL sTNFR for 8 h in the microwell device (four conditions). Data colored by relative secretion intensity of the indicated protein (A) or cell density (B) for the indicated subpopulation of cells (other cells grayed out for visualization). (C) KDE for BMDMs along the PHATE 1 axis stimulated as indicated and calculated from data shown in (A) and (B). Arrows indicate intermediate and high modes of the trimodal PHATE 1 distribution. Number of biological replicates vary by experiment: LPS alone (n = 3); sTNFR (n = 2); IFNAR and IL-10R (n = 1). (D) Bar graphs of percentage of cells secreting the indicated protein above threshold in response to the indicated cues after 8 h in the microwell assay. Data presented as mean ± 95% CIs calculated by bootstrapping. Significance determined by non-overlap of CIs. (E) Dose response of TNF secretion following LPS stimulation of BMDMs for 24 h. TNF secretion was measured by ELISA and presented as mean ± SEM of 2 biological replicates. (F) Schematic model illustrating dual roles for IL-10 in the TLR4 response.