Fig 1.
Multiple signaling axes regulate macrophage phenotype and polarization.
Macrophage polarization is dynamically controlled by different receptor-mediated signaling pathways, cell stress, transcriptional and post-transcriptional regulators (e.g. miRs), which collectively lead to differential expression of a panel of macrophage phenotype markers (including both intracellular and secreted proteins). Arrow indicates activation,–| symbol indicates inhibition. Green shapes indicate intracellular proteins, orange shapes indicate secreted products. This figure is an overview of model formulation; full mechanistic details of the computational model are presented in S1 Fig and S1 and S2 Tables. It should also be noted that this figure only describes a subset of the M1- and M2-related pathways and markers.
Fig 2.
IFN-γ-mediated signaling controls macrophage phenotype.
In response to different doses of IFN-γ treatment, the model simulations are compared with corresponding literature time-course data including (A) degradation of receptor-bound IFN-γ [59], (B) phosphorylation of receptor-associated JAK [15], (C) phosphorylation of STAT1 [60], (D) expression of IRF-1 [61], (E) expression of iNOS [62], (F) levels of secreted TNFα [17], (G) levels of secreted IL-12 [16], (H) levels of secreted CXCL-9 [16], (I) intracellular mRNA expression of CXCL-10 [63], (K) expression of miR-3473b [42], plus single timepoint measurements including intracellular levels of (J) itaconic acid at 18 h [64], (L) PTEN at 36 h (in response to miR-3473b mimic transfection, see also S2H Fig) [42], and (M) HIF-1α (in response to IFN-γ with or without hypoxia) [48]. (A-M) All literature data are measured in macrophage cell lines and values are for protein levels unless noted otherwise. Y-axes show normalized expression respectively (A-E, G-I, K: simulations and data are normalized to the maximum expression; F, L: normalized to the no-treatment/time 0 expression; J: normalized to the expression at 18 h; M: normalized to the expression under IFN-γ treatment with hypoxia). S–simulation, D–literature data, Utr–untreated, Trd–IFN-γ treated, Hyp–hypoxia.
Fig 3.
IL-4-mediated signaling controls macrophage phenotype.
Comparison between model simulations and literature experimental data on IL-4 induced (A) STAT6 phosphorylation [65], (B-C) IRF-4 upregulation (time-course and at 24 h) [66, 67], (D) AKT activation [68], (E) PPARγ expression at 18 h [69, 70], (F-G) Arg-1 expression (time-course and at 24 h) [67, 71], (H) IL-10 secretion at 24 h [72], (I) VEGF secretion at 24 h [73], (J) downregulation of TNFα secretion at 24 h [72], and (K) HIF-2α stabilization (in response to IL-4 with or without hypoxia) [48]. (A-K) All literature data are measured in macrophage cell lines and values are for protein levels unless noted otherwise. Y-axes show normalized expression respectively (A, B, D, F: simulations and data are normalized to the maximum expression; C, G: normalized to the expression at 24 h post-treatment; E, H, I, J: normalized to the no-treatment/time 0 expression; K: normalized to the expression under IL-4 treatment with hypoxia). S–simulation, D–literature data, Utr–untreated, Trd–IL-4 treated, Hyp–hypoxia.
Fig 4.
Hypoxia promotes M1 and M2 marker expression.
Model simulation and literature experimental data from macrophages on hypoxia-induced (A) time-course stabilization of HIF-1α and (B) HIF-2α under 3% O2 [75, 76], (C) sustained stabilization of HIF-1/2α at 24 h under 0.5% O2 [77], (D) upregulation of iNOS and (E) Arg-1 proteins at 8 h under 1% O2 [78], (F) increase in TNFα secretion at 24 h under 0.3% O2 [79], (G) increase in IFN-γ secretion over time under 1% O2 [47], (H) increase in VEGF secretion at 24 h under 1% O2 [80], and (I) inhibition of miR-93 abundance at 12 h under 2% O2 [53]. (J) Enforced overexpression of miR-93 (see also S4G Fig) leads to decreased IFN-γ secretion at 12 h under 2% O2 [53]. (A-J) All literature data are measured in macrophage cell lines and results are for protein levels unless noted otherwise. Y-axes show normalized expression respectively (A, B: simulations and data are normalized to the maximum expression; C: normalized to the expression at 24 h under hypoxia; D-I: normalized to the normoxic/time 0 expression; J: normalized to the hypoxia-induced expression at 12 h without miR-93 mimic treatment). S–simulation, D–literature data, Utr–normoxia/untreated, Trd–treated with miR-93 mimic, Hyp–hypoxia, O2 –oxygen.
Fig 5.
Pathway feedbacks and cross-talks in M1-M2 regulatory network.
Overexpression of SOCS1 and SOCS3 in macrophages can downregulate activation of (A) STAT1 by IFN-γ and (B) STAT6 by IL-4. Silencing of SOCS3 promotes (C) IFN-γ-induced M1 marker expression while it minimally affects (D) IL-4-induced M2 marker expression (relative fold changes are labeled). (A-D) Overexpression is modeled as 50x initial level with normal (1x) production, and silencing is modeled as 0 initial level with 0 production. (E) Simulated dose response of iNOS and Arg-1; relative protein levels measured at 12 h are plotted and labeled (the baseline condition is represented by the 0.01 ng/ml case). (F) Upon IFN-γ stimulation followed by the addition of IL-4 (at 4 h), cellular IRF-1 level is downregulated compared to IFN-γ only; (G) Upon IL-4 stimulation followed by the addition of IFN-γ (at 1 hr), cellular activation of AKT is downregulated compared to IL-4 only. (H) The addition of a second stimulus IFN-γ (after 24 h of IL-4 stimulation) would antagonize the expression pattern of M1 and M2 markers induced by IL-4 (see also S5G Fig). (I) Similarly, IL-4 added after 24 h of IFN-γ stimulation would antagonize the marker expression pattern induced by IFN-γ. When macrophages are stimulated with IFN-γ and IL-4 simultaneously, the simulated expression of (J) M1 and M2 markers as well as (K) the activation of a number of M1 and M2 signature proteins (see also S5I Fig) are collectively induced with distinct temporal profiles. (L) Dynamic protein expression patterns (after 12, 24 and 48 h of stimulation) of M1 and M2 markers in macrophages under seven different stimulation conditions (A+B means simultaneous stimulation, expression levels are normalized to the untreated/time 0 levels and then log2 transformed). (A-L) All simulation results are protein levels (except CXCL10 is mRNA level). (C-E, H-K) Y-axes show relative expression respectively (C-D, H-K: normalized to untreated/control/time 0 levels; E: normalized to maximum levels at 50 ng/ml). Simulated treatment doses are 10 ng/ml IFN-γ and 10 ng/ml IL-4 for (A-D), 10 ng/ml IFN-γ and 20 ng/ml IL-4 for (F-G), 20 ng/ml IFN-γ and 20 ng/ml IL-4 for (H-I), 10 ng/ml IFN-γ and 5 ng/ml IL-4 for (J-L). Utr–untreated, hyp–hypoxia (2% oxygen for L).
Fig 6.
Global sensitivity analysis and simulated therapeutic strategies to repolarize macrophages in hypoxia.
(A-B) Sensitivity indices (top 25 positive and negative PRCC values with p<0.05) of model parameters that control M1 and M2 marker expression in terms of the M1/M2 score (a ratio-based estimate of M1 phenotypes relative to M2 phenotypes, see Materials and Methods for more details) in hypoxia (2% O2). In the parameter descriptions, ‘X_RC’ means receptor complex formed by ligand X, receptor and JAK, ‘X/Y’ means complex formed by X and Y. Simulated time-course expression of M1 and M2 markers when macrophages are subjected to (C-D) hypoxia, (E-F) hypoxia with IFN-γ inhibition, (G-H) hypoxia with HIF-1α inhibition, (I-K) hypoxia with STAT1 inhibition, and (L-M) hypoxia with IRF-1 inhibition. Inhibition of IFN-γ, HIF-1α and IRF-1 is simulated by setting the respective production rates to 10% of their original values (STAT1 inhibition is simulated as a 90% decrease in the binding rate between STAT1 and activated IFN-γ receptor complex). Species name denoted with * means expression in hypoxia plus treatment (species name without * means expression in hypoxia alone). (C-M) Marker expression levels are normalized to their respective t = 0 values (e.g. normoxia, unstimulated). (A-B) More details about the parameters listed can be found in S1 Table using the labels (positive–k127, kf63, kr70, kf64, kf17, k33, k61, k77, k45, kf44, kf42, k37; negative–k99, kr42, k78, kf8, kr44, kf95, k71, kf13, ka77, kf7, kf52, kf70, kr64; order is from top to bottom as displayed). (C-M) All simulation results are protein levels (except CXCL10 is mRNA level).
Fig 7.
Targeting IL-4 signaling axis in macrophages in tumor.
Simulated time-course expression of M1 and M2 markers when macrophages are subjected to (A-B) high IL-4 production (10x of original value), (C-D) high IL-4 production with IL-4/receptor blockade (90% decrease in the binding rate between IL-4 and its receptor), (E-F) high IL-4 production with STAT6 inhibition (90% decrease in the binding rate between STAT6 and activated IL-4 receptor complex), and (G-H) high IL-4 production with PHD inhibition (90% decrease in the binding rate between PHD and O2). Species name denoted with * means expression in high IL-4 production plus treatment (species name without * means expression in high IL-4 production alone). (A-H) Marker expression levels are normalized to their respective t = 0 values (e.g. normoxia, unstimulated). All simulation results are protein levels (except CXCL10 is mRNA level).