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. 2016 Jul 18;126(8):3089–3103. doi: 10.1172/JCI83404

Figure 3. P21–/– macrophages show impaired ability to polarize to M2 cells during in vitro endotoxin tolerance.

Figure 3

(A) Scheme showing the in vitro endotoxin tolerance model. Peritoneal macrophages from WT and P21–/– mice were tolerized with 100 ng/ml LPS for 20 hours (Tol), washed with PBS, cultured in medium (2 hours), and restimulated with 100 ng/ml LPS for 4 hours (Tol + LPS). LPS-activated cells were stimulated with LPS for 4 hours without previous tolerization (LPS). Cells left untreated were controls (Ctrl). Total RNA was extracted at 4 hours after LPS treatment and analyzed for gene expression. Culture supernatants were analyzed for cytokine production. (B) RT-PCR analysis showed impaired upregulation of M2-associated genes in P21–/– compared with WT macrophages after tolerization. (C) RT-PCR showed upregulation of representative M1 cytokine genes in P21–/– compared with WT tolerized macrophages. Results were normalized to β-actin and represent fold induction over unstimulated WT cells. (D) M1-associated TNF-α and IFN-β and M2-associated CCL17 production in WT and P21–/– macrophages at different time points after LPS restimulation, as measured by ELISA. In all cases data show the mean ± SEM (n = 3 independent experiments), *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed Student’s t test.