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 5

p21 modulates the balance between p65-p50 and p50-p50 NF-κB dimers in LPS-tolerized macrophages.

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p21 modulates the balance between p65-p50 and p50-p50 NF-κB dimers in LP...
WT and P21–/– resting or tolerized (20 hours) peritoneal macrophages were stimulated with LPS (4 hours). NF-κB complexes that bound the consensus or the Ifnb promoter sequence were analyzed by EMSA. Anti–p50 and –p65 NF-κB antibodies were used for supershift analysis. Supershift intensity was assessed by densitometry and plotted as the percentage of supershifted complex at indicated times after LPS stimulation. (A) Left, Increased NF-κB binding to the consensus sequence in P21–/– (lane 5) compared with WT (lane 2) LPS-activated macrophages (shown at 15 minutes). Right, Relative NF-κB complex composition at different times after LPS activation. (B) Left, Similar NF-κB binding to the consensus sequence in WT (lane 2) and P21–/– (lane 5) LPS-tolerized macrophages (shown at 15 minutes) and reduced p50-p50 DNA binding in P21–/– macrophages as indicated by arrows (compare lanes 4 and 7). Right, Delayed switch to p50-p50 NF-κB complex composition in P21–/– LPS-tolerized macrophages. (C) Left, Similar NF-κB binding to the Ifnb promoter sequence in WT (lane 2) and P21–/– (lane 5) macrophages after LPS restimulation (shown at 30 minutes) and reduced p50-p50 DNA binding in P21–/– macrophages (compare lanes 4 and 7; arrow). Right, Delayed switch to p50-p50 NF-κB complexes bound to the Ifnb promoter sequence in P21–/– LPS-tolerized macrophages. In all gels, the first lane is nuclear extract of untreated macrophages (negative control). Representative results of 2 independent experiments are shown.