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. 2009 Sep 1;106(35):14978-83.
doi: 10.1073/pnas.0809784106. Epub 2009 Aug 17.

Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor kappaB

Chiara Porta  1 , Monica Rimoldi, Geert Raes, Lea Brys, Pietro Ghezzi, Diana Di Liberto, Francesco Dieli, Serena Ghisletti, Gioacchino Natoli, Patrick De Baetselier, Alberto Mantovani, Antonio Sica
Affiliations

Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor kappaB

Chiara Porta et al. Proc Natl Acad Sci U S A. .

Abstract

Cells of the monocyte-macrophage lineage play a central role in the orchestration and resolution of inflammation. Plasticity is a hallmark of mononuclear phagocytes, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic or M1 and alternative or M2. NF-kappaB is a key regulator of inflammation and resolution, and its activation is subject to multiple levels of regulation, including inhibitory, which finely tune macrophage functions. Here we identify the p50 subunit of NF-kappaB as a key regulator of M2-driven inflammatory reactions in vitro and in vivo. p50 NF-kappaB inhibits NF-kappaB-driven, M1-polarizing, IFN-beta production. Accordingly, p50-deficient mice show exacerbated M1-driven inflammation and defective capacity to mount allergy and helminth-driven M2-polarized inflammatory reactions. Thus, NF-kappaB p50 is a key component in the orchestration of M2-driven inflammatory reactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
LPS-tolerant monocytes express an M2 cytokine/chemokine profile. Total RNA from control (M/M), activated (M/L), tolerant (L/M), and tolerant monocytes rechallenged with LPS (L/L) were analyzed by RT-PCR for the expression of representative M1 genes (filled bars) and M2 genes (open bars). Results are given as fold increase over the mRNA level expressed by untreated cells (M/M) and are representative of at least 3 different experiments; shown are mean ± SD from triplicate values. For ELISA, results are the average of 3 independent experiments ± SD. ***P < 0.001, t test.
Fig. 2.
Fig. 2.
Role of p50 NF-κB in macrophage polarization. (A) peritoneal macrophages from WT (filled bars) and p50−/− (open bars) mice were either untreated (M/M), activated 4 h with LPS (M/L), or tolerized without (L/M) or with LPS rechallenge (L/L). Representative M1 and M2 cytokine and chemokine genes were analyzed by RT-PCR (A) and chromatin immunoprecipitation (ChIP) of RNA Pol II (B) on representative M1 (TNFα, iNOS, and IFNβ) and M2 (CCL17 and Arginase I) genes, as well as of the NF-κB subunits p50 (open bars) and p65 (filled bars) on the IL-10 gene promoter (C). Results were normalized to β-actin level and expressed as fold induction with respect to the control cell population. Data are representative of 3 independent experiments; shown are mean ± SD from triplicate values. **P < 0.01; ***P < 0.001, t test.
Fig. 3.
Fig. 3.
p50 NF-κB acts as negative regulator of IFN-β production and STAT1 activity. PEC were isolated from WT (black bars), p50−/− (white bars), IRF-3−/− (dark gray bars) and double p50/IRF-3−/− (light gray bars) mice, as indicated. Next, cells were activated with LPS (100 ng/mL) and analyzed for IFN-β expression (A) and cytokine expression (A and B). Total RNA was extracted 4 h after LPS treatment (100 ng/mL) and analyzed by RT-PCR. Supernatants were collected 24 h after LPS and tested by ELISA for IFN-β. (C) RAW 267.4 cells were cotransfected with 0.5 μg of the luciferase reporter plasmid containing the murine IFN-β promoter region −125 to +55 and increasing amounts of a CMV-driven p50 NF-κB expression vector, as indicated. Luciferase activity is expressed as fold induction with respect to untreated cells. Results shown are the average ± SD of 3 independent experiments. **P < 0.01, t test. ***, P < 0.001. (D) EMSA analysis of the NF-κB complexes binding the IFN-β promoter region −64 to −55 (28) in PEC untreated (M/M), M1-activated (M/L), and LPS-tolerant (L/L). Antisera against the p50 and p65 NF-κB subunits were used for supershift analysis, as indicated. (E) Peritoneal macrophages were isolated from WT, p50−/−, IRF-3−/−, and double p50/IRF-3−/− mice and stimulated with LPS for 90 min. Next STAT1 phosphorylation was analyzed by Western blot. Equal loading is visualized by actin expression.
Fig. 4.
Fig. 4.
Essential role of p50 NF-κB in the development of M2-polarized inflammation associated with chronic T. crassiceps infection. WT and p50−/− mice were i.p. inoculated with 25 nonbudding T. crassiceps metacestodes. Expression of M1 and M2 genes by WT (filled bars) and p50−/− (open bars) peritoneal macrophages was analyzed in naïve, early-stage infected (<4 weeks), and late-stage infected (>6 weeks) mice. Data are normalized to actin gene expression and shown as fold increase in mRNA expression with respect to WT naïve macrophages. At each time point, the results shown are the mean ± SEM of 4 naïve and infected mice. Parasite loads were evaluated at 8 weeks after infection. **P < 0.01; ***P < 0.001, t test, mean ± SEM, n = 4.
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