The model accurately reproduces activation of inflammatory and anti-apoptotic signals, controlled through IL-1RI and TILRR.
A. TILRR siRNA decreases IκBα degradation in HeLa cells transfected with IκBα-EGFP (7μg/106 cells) alone or in the presence of random (♦) or TILRR-specific (■) siRNA and stimulated with IL-1 (1 nM). Graph show continuous readings from three experiments including 55 cells, p<0.05 at 60 min. B. Simulations using the best-fit model, show reductions in transfected IκBα-EGFP degradation during IL-1β stimulation in the presence (Red) and absence (Blue) of TILRR, and are in agreement with published data (A). C, D. TILRR cDNA increases (C) and TILRR siRNA decreases (D) IL-8 luciferase activity in HeLa cells treated with IL-1β (1nM, 6 hrs.) (Mean±SEM, n = 3, *p <0.05; **p< 0.01). E, F. Simulations of un-stimulated levels (black) and IL-1β-induced IL-8 activity (blue) in the presence of the IL-1RI complex and with increasing (E) and decreasing (F) TILRR expression relative to endogenous levels, using the agent-based model, show agreement with in vitro data in C and D. (Mean±SEM, n = 3, 0.005In vitro experiments (G) (Mean± SEM, n = 3, * p <0.005) and model simulation (H) (Mean ± SEM, n = 3, WT vs. D448 p<0.0.001). I, J. Mutation of residue D448 results in a 60% reduction in inflammatory gene activity, while other mutants have no impact (I) (arrows indicate WT TILRR and the R425 and D448 mutants, Mean±SEM, n = 3, * p<0.001). Model simulations (J) similarly show a pronounced reduction in the presence of the D448 mutant, but no effect by substitution of R425. (Mean±SEM, n = 3, WT vs. D448 p<0.001). K, L. TILRR-induced increase in levels of phosphor-Akt by IL-1β (1nM) is blocked following mutation of residue R425, while substitution at D448 has the same effect as the wild type co-receptor. In vitro experiments (K) (Data are expressed relative to levels in mock transfected cells, Mean±SEM, n = 3, WT vs. R425, p<0.01), and model simulation (L) (Mean±SEM, n = 3, wt vs. R425, p<0.005). M, N. Mutation of TILRR residue R425 blocks anti-apoptotic responses, determined by TUNEL staining in in vitro experiments, while the D448 substitution has no impact (M) (Mean ±SEM, n = 3, * p<0.001.). Model simulations show that TILRR-induced reductions in pro-apoptotic Caspase-3, is blocked by the R425 mutation but unaffected by substitution at D448 (N) (Mean±SEM, n = 3, wt vs. R425, p<0.01). Graphs in A, C, D, G, I, K, M are reproduced from Zhang, et al. [13,14], and used for validation of the model and for comparison with agent based model output.