Deciphering signaling outcomes from a system of complex networks

Abstract

Cellular signal transduction machinery integrates information from multiple inputs to actuate discrete cellular behaviors. Interaction complexity exists when an input modulates the output behavior that results from other inputs. To address whether this machinery is iteratively complex--that is, whether increasing numbers of inputs produce exponential increases in discrete cellular behaviors--we examined the modulated secretion of six cytokines from macrophages in response to up to five-way combinations of an agonist of Toll-like receptor 4, three cytokines, and conditions that activated the cyclic adenosine monophosphate pathway. Although all of the selected ligands showed synergy in paired combinations, few examples of nonadditive outputs were found in response to higher-order combinations. This suggests that most potential interactions are not realized and that unique cellular responses are limited to discrete subsets of ligands and pathways that enhance specific cellular functions.

Fig. 1

Secretion of cytokines in response to single ligands. RAW 264.7 cells were treated with KDO (black circle), IFN-β (green diamond), IL-6 (light-blue triangle), ISO (red square), 8Br-cAMP (dark-blue hexagon), TGF-β (pink circle), or were left untreated (open inverted triangle) for 150, 195, or 240 minutes. The supernatants were removed, and the concentrations of IL-10, IL-6, G-CSF, TNF-α, RANTES, and MIP-1α were measured (pg/ml). The normalized traces [mean ± standard deviation (s.d.), n>10) are shown. For traces in which the lines overlap, additional staggered symbols are shown for easy identification.

Fig. 2

Classification of interactions between ligands. Two ligands, L1 and L2, are capable of eliciting cytokine secretion individually (A, solid black lines). When applied in combination, the two ligands may act in any one of several ways: they may act independently of each other yielding an additive response (dotted black line, surrounding white region); they could synergize to yield a greater-than-additive response (red); they may inhibit each other to yield a response lower than the individual responses (blue); they could elicit some intermediate response between the responses of the individual ligands and that of the predicted additivity (yellow to green). The last set of responses may be mechanistically generated by any one of several processes, including saturation and noncompetitive antagonism, among others. (B) The significance of nonadditivity can be calculated and displayed by this colorimetric scheme in a heat map.

Fig. 3

Secretion of IL-10 in response to KDO, IFN-β, and IL-6. RAW 264.7 cells were treated with single, double, or triple combinations of ligands. The results for each cytokine were normalized and the statistical significance was determined as described in the Materials and Methods. Examples of secretion of IL-10 are shown for the dual (A and B) and triple (D) combinations of ligands. (C) Secretion of IL-6 can also be synergistically induced by the combination of KDO with IFN-β. Panels A to C display the single ligand responses, the predicted response of combinations of ligands (expected), and the observed values for combinations (observed). Panel D shows only the predicted and observed values for the 3-way combinations. Statistical significance for nonadditivity of the combinations is shown at the right.

Fig. 4

Secretion of MIP-1α in response to KDO, IFN-β, and IL-6. RAW 264.7 cells were treated with single, double, or triple combinations of ligands and the secretion of MIP-1α was measured as described. The results from multiple experiments were normalized and statistical significance was determined as described in the Materials and Methods. Each panel shows the single ligand responses and the observed and predicted (expected) responses for dual (A and B) and triple (C) ligand combinations. The predicted response is based on the additive effects of the ligands; the bar underneath the graphs quantitates the nonadditivity of the ligand combinations.

Fig. 5

Secretion of G-CSF in response to KDO, IFN-β, and ISO. The secretion of G-CSF (A and B) by RAW 264.7 cells was measured in response to KDO, IFN-β, and either ISO or 8Br. (C) The relative abundance of G-CSF mRNA was quantified by qRT-PCR. A single representative experiment is shown. Error bars show the range between duplicate samples. All samples were normalized to time-matched untreated samples. All columns except the responses to IFN-β alone were significantly different from the untreated sample (p<0.05).

Fig. 6

Secretion of MIP-1α in response to KDO, IFN-β, and ISO. The secretion of MIP-1α by RAW 264.7 cells was measured in response to KDO, IFN-β, and either ISO (A) or 8Br (B). Results are the average of 2 to 5 normalized experiments each containing duplicate samples. (C) The relative abundance of G-CSF mRNA was quantified by qRT-PCR. A single representative experiment is shown. Error bars show the range between duplicate samples. All samples were normalized to time-matched untreated samples. All columns were statistically significantly different from the untreated sample (p<0.05).

Fig. 7

Secretion of IL-6 in response to KDO, IFN-β, and ISO. The secretion of IL-6 by RAW 264.7 cells was measured in response to KDO, IFN-β, and ISO (A) or 8Br (B). The average of the normalized values of 2 to 5 experiments, each with at least duplicate samples, are shown.

Fig. 8

Secretion of IL-6 in response to KDO, IFN-β, TGF-β, and the cAMP pathway. The secretion of IL-6 by RAW 264.7 cells was measured after treatment of cells with KDO, IFN-β, TGF-β, and either ISO or 8Br for the indicated times. The normalized values of 2 to 5 experiments with samples prepared in at least duplicate were averaged and the errors determined.

Fig. 9

Quantified, synergistic cytokine responses to stimulation with multiple ligands. A summary matrix of the cytokine responses in RAW 264.7 cells is shown for all treatments performed with single, double, triple, quadruple, and quintuple combinations of ligands. The results for each cytokine were normalized and the statistical significance was determined as described in the Materials and Methods.