p21 mediates macrophage reprogramming through regulation of p50-p50 NF-κB and IFN-β
Gorjana Rackov, … , Carlos Martínez-A, Dimitrios Balomenos
Gorjana Rackov, … , Carlos Martínez-A, Dimitrios Balomenos
Published July 18, 2016
Citation Information: J Clin Invest. 2016;126(8):3089-3103. https://doi.org/10.1172/JCI83404.
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Research Article Immunology

p21 mediates macrophage reprogramming through regulation of p50-p50 NF-κB and IFN-β

Abstract

M1 and M2 macrophage phenotypes, which mediate proinflammatory and antiinflammatory functions, respectively, represent the extremes of immunoregulatory plasticity in the macrophage population. This plasticity can also result in intermediate macrophage states that support a balance between these opposing functions. In sepsis, M1 macrophages can compensate for hyperinflammation by acquiring an M2-like immunosuppressed status that increases the risk of secondary infection and death. The M1 to M2 macrophage reprogramming that develops during LPS tolerance resembles the pathological antiinflammatory response to sepsis. Here, we determined that p21 regulates macrophage reprogramming by shifting the balance between active p65-p50 and inhibitory p50-p50 NF-κB pathways. p21 deficiency reduced the DNA-binding affinity of the p50-p50 homodimer in LPS-primed and -rechallenged macrophages, impairing their ability to attenuate IFN-β production and acquire an M2-like hyporesponsive status. High p21 levels in sepsis patients correlated with low IFN-β expression, and p21 knockdown in human monocytes corroborated its role in IFN-β regulation. The data demonstrate that p21 adjusts the equilibrium between p65-p50 and p50-p50 NF-κB pathways to mediate macrophage plasticity in LPS tolerance. Identifying p21-related pathways involved in monocyte reprogramming may lead to potential targets for sepsis treatment.

Authors

Gorjana Rackov, Enrique Hernández-Jiménez, Rahman Shokri, Lorena Carmona-Rodríguez, Santos Mañes, Melchor Álvarez-Mon, Eduardo López-Collazo, Carlos Martínez-A, Dimitrios Balomenos

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Figure 3

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

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P21–/– macrophages show impaired ability to polarize to M2 cells during...
(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.