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 7

p21 regulates hyporesponsiveness of human monocytes from healthy donors and sepsis patients.

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p21 regulates hyporesponsiveness of human monocytes from healthy donors ...
(A) Total RNA was extracted from monocytes isolated from sepsis patients and healthy volunteers and analyzed by RT-PCR. Top, P21 mRNA levels were significantly higher in monocytes from sepsis patients than those from controls. The median is shown (n = 7), *P < 0.05, 2-tailed Mann-Whitney U test. Bottom, Correlation between P21 and Ifnb levels in monocytes from sepsis patients. Results were normalized to β-actin and log2 values were used to calculate correlation; n = 7, 2-tailed Pearson’s test r = –0.7944, *P < 0.05. (B) Monocytes from healthy volunteers and sepsis patients were challenged ex vivo with 10 ng/ml LPS (1 hour). RT-PCR analysis showed Ifnb upregulation in monocytes from healthy donors but not from sepsis patients (top). High P21 expression was detected in monocytes from sepsis patients before and after LPS treatment (bottom); (n = 5), 1-tailed Student’s t test. NS, not significant. (C) Cultured human monocytes from healthy volunteers were tolerized with LPS, washed, and restimulated with LPS. RT-PCR analysis showed high P21 expression in tolerant human monocytes (top). Ifnb expression was downregulated in tolerant cells; (n = 4), 1-way ANOVA. (D) Cultured human monocytes from healthy volunteers were transfected with p21 siRNA or control siRNA. Cells were then LPS tolerized and rechallenged with LPS. After LPS rechallenge, RT-PCR showed that P21 mRNA was abolished in cultures transfected with P21-specific siRNA, and Ifnb expression was upregulated compared with control siRNA–transfected cells; (n = 4), 2-tailed Student’s t test. In all RT-PCR experiments, results were normalized to β-actin. Data shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.