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 6

p21 regulates p50-p50 NF-κB DNA binding.

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p21 regulates p50-p50 NF-κB DNA binding. (A) RT-PCR analysis showing sim...
(A) RT-PCR analysis showing similar Nfkb1 (encoding p105) and Bcl3 gene expression in WT and P21–/– LPS-tolerized/restimulated peritoneal macrophages. Results were normalized to β‑actin and show fold induction over unstimulated WT cells. Data are representative of 3 independent experiments. (B) Immunoblot showing similar p50 protein levels in the cytoplasmic and nuclear fractions of WT and P21–/– LPS-tolerized/restimulated peritoneal macrophages. β-Actin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. (C) Reduced ubiquitination of p50 in P21–/– LPS-tolerized/restimulated peritoneal macrophages. Equal amounts of protein were immunoprecipitated with antibody against p50 and immunoblotted with antibody against ubiquitin. n.c., negative control; IP performed in the absence of cell extracts. (D) Reduced p50 phosphorylation in P21–/– LPS-tolerized/restimulated peritoneal macrophages. Equal amounts of protein were immunoprecipitated with antibody against p50 and immunoblotted with antibody against phospho-serine/threonine. (E) p21 increases p50 homodimer binding to DNA by increasing its affinity. Tolerized (20 hours) WT and P21–/– peritoneal macrophages were stimulated with LPS (15 minutes). Anti–p50 NF-κB antibody was used for supershift assays with increasing amounts of unlabeled oligonucleotide (cold probe), and NF-κB complexes binding the Ifnb promoter sequence were analyzed by EMSA. (F) p50-p50 and p65-p50 DNA binding was measured by densitometry of autoradiogram in E. Shown are representative gels of at least 2 experiments performed.