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 4

IFN-β neutralization reduces STAT1 phosphorylation and induces hyporesponsiveness in P21–/– macrophages.

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IFN-β neutralization reduces STAT1 phosphorylation and induces hyporespo...
(A) P21 mRNA and protein levels in LPS-rechallenged WT macrophages. (B) STAT1 phosphorylation in WT and P21–/– macrophages. (C) Increased iNOS and CXCL11 expression in tolerized P21–/– macrophages. (D and E) P21–/– peritoneal macrophages were incubated with an IFN-β– or TNF-α–neutralizing antibody or an isotype control during LPS tolerization (20 hours). Cells were washed, cultured in medium (2 hours) and restimulated with LPS (4 hours). (D) Reduction in STAT1 phosphorylation and iNOS expression after antibody treatment at indicated times. (E) RT-PCR analysis of P21–/– macrophages incubated with appropriate antibodies during LPS tolerization (20 hours) and restimulated with LPS (4 hours). (F) After LPS tolerization, P21–/– macrophages were restimulated with LPS (4 hours) in the presence of anti–IFN-β or isotype control antibody. Immunoblot shows a reduction in STAT1 phosphorylation and iNOS expression. (G) WT macrophages were treated with IFN-β during LPS tolerization, restimulated with LPS (4 hours), and analyzed by RT-PCR. (H) P21–/– mice were challenged with 2 LPS doses as in Figure 1. At 2 hours before LPS rechallenge, mice were treated i.p. with anti-IFNAR1 antibody or control IgG. Inhibition of IFNAR1 improved tolerance to LPS, as shown by a Kaplan-Meier survival curve (n = 9), ***P < 0.001, log‑rank (Mantel-Cox) test. For all RT-PCR analyses, results were normalized to β‑actin and show fold induction over unstimulated WT cells. Data shown as the mean ± SEM (n = 3 independent experiments), *P < 0.05, **P < 0.01, ***P < 0.001. For A and E, 1-way ANOVA. For C and G, 2-tailed Student’s t test. NS, not significant. For immunoblots, β-actin was used as a loading control. Western blots in A and B were derived from the same experiment. Representative gels of 3 experiments are shown.