Figure 1: TILRR Controls IL-1RI Levels, Signal Amplification, and Gene Activity

(A) Quantitative polymerase chain reaction (qPCR) of TILRR expression in raw cells, stimulated with lipopolysaccharide (LPS) (6 h) over a range of concentrations, as indicated. Data are expressed relative to levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and show mean ± SEM. n = 3.0 μg/ml LPS versus 0.1μg/ml LPS; 0 μg/ml LPS versus 1 μg/ml LPS, **p < 0.01. (B) qPCR of Toll-like and interleukin-1 receptor regulator (TILRR) expression in M1- and M2-like macrophages and undifferentiated cultures (control). Data are expressed relative to levels of GAPDH and show mean ± SEM. n = 3, **p < 0.01,***p < 0.001. (C) Fluorescence-activated cell sorting analysis of inflammatory monocytes from wild-type (WT) and TILRR knockout (KO) mice injected with LPS (10 mg/kg). Data are expressed as percent of total monocyte levels and show mean ± SEM. n = 6 WT, n = 9 TILRR KO. Levels in TILRR KO mice versus levels in WT mice, **p < 0.0088. (D) Western blots of interleukin-1 receptor type I (IL-1RI), tumor necrosis factor receptor (TNFR), Toll-like receptor 4 (TLR4), and Toll-like receptor 2 (TLR2) expressions in spleen from WT and TILRR KO mice. (E) Quantitation of Western blots as in D showing receptor levels in TILRR KO cells relative to levels in WT cells, using to β-actin as loading control. The graph shows mean ± SEM, n = cells from 4 to 6 WT mice and 4 to 6 TILRR KO mice for each receptor. Levels of IL-1RI, TNFR, TLR4, and TLR2 in TILRR KO mice are expressed as percent of levels of the respective receptor in WT mice, IL-1RI expression in TILRR KO mice versus IL-1RI expression in WT mice. ****p < 0.0001. (F) Western blot analysis of interleukin (IL)-1–induced (10−9 M) inhibitor kappa B alpha (IκBα) degradation in bone marrow–derived macrophages from WT and TILRR KO mice. (G) Quantitation of Western blots as in F showing IL-1–induced IκBα degradation in WT (circles) and TILRR KO (squares) cells. Data are expressed as percent of IκBα levels in unstimulated WT and TILRR knockout cells, respectively (time 0) and show mean ± SEM. n = peripheral blood mononuclear cells from 4 WT mice and 4 TILRR KO mice. Levels in TILRR KO cells versus levels in WT cells at 30 min. *p = 0.0124. (H) qPCR of bone marrow–derived macrophages from TILRR KO mice shows reductions in chemokine ligand 2 (CCL2) and C-X3-C motif chemokine ligand 1 (CX3CL1). Data are expressed as percent activation of the respective gene in cells from WT mice and show mean ± SEM. n = cells from 4 TILRR KO mice and 4 WT mice. CCL2 expression in TILRR KO cells versus expression in WT cells, **p = 0.0022; CX3CL1 expression in TILRR KO cells versus expression in WT cells, *p = 0.0482. (I) qPCR of spleen samples from TILRR KO mice shows reductions in proinflammatory cytokines tumor necrosis factor (TNF)-α and IL-6. Data are expressed as percent activation of the respective gene in spleen samples from WT mice, and show mean ± SEM. n = 5 WT and 5 TILRR KO mice. TNF-α expression in TILRR KO cells versus expression in WT cells, ***p = 0.0009. IL-6 expression in TILRR KO cells versus expression in WT cells, *p = 0.0236.