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. 2016 Jun 10;291(24):12851-12861.
doi: 10.1074/jbc.M116.718825. Epub 2016 Apr 18.

The Atypical Inhibitor of NF-κB, IκBζ, Controls Macrophage Interleukin-10 Expression

Sebastian Hörber  1 , Dominic G Hildebrand  1 , Wolfgang S Lieb  1 , Sebastian Lorscheid  1 , Stephan Hailfinger  1 , Klaus Schulze-Osthoff  2 , Frank Essmann  3
Affiliations

The Atypical Inhibitor of NF-κB, IκBζ, Controls Macrophage Interleukin-10 Expression

Sebastian Hörber et al. J Biol Chem. .

Abstract

Macrophages constitute a first line of pathogen defense by triggering a number of inflammatory responses and the secretion of various pro-inflammatory cytokines. Recently, we and others found that IκBζ, an atypical IκB family member and transcriptional coactivator of selected NF-κB target genes, is essential for macrophage expression of a subset of pro-inflammatory cytokines, such as IL-6, IL-12, and CCL2. Despite defective pro-inflammatory cytokine expression, however, IκBζ-deficient mice develop symptoms of chronic inflammation. To elucidate this discrepancy, we analyzed a regulatory role of IκBζ for the expression of anti-inflammatory cytokines and identified IκBζ as an essential activator of IL-10 expression. LPS-challenged peritoneal and bone marrow-derived macrophages from IκBζ-deficient mice revealed strongly decreased transcription and secretion of IL-10 compared with wild-type mice. Moreover, ectopic expression of IκBζ was sufficient to stimulate Il10 transcription. On the molecular level, IκBζ directly activated the Il10 promoter at a proximal κB site and was required for the transcription-enhancing trimethylation of histone 3 at lysine 4. Together, our findings show for the first time the IκBζ-dependent expression of an anti-inflammatory cytokine that is crucial in controlling immune responses.

Keywords: NF-κB; NF-κB transcription factor; immunosuppressor; interleukin; macrophage.

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Figures

FIGURE 1.
FIGURE 1.
IκBζ is essential for Il10 expression in mouse embryonic fibroblasts and peritoneal macrophages. A, MEFs were cultured in the presence of 1 μg/ml LPS for 5 h. Total RNA was isolated and subjected to qRT-PCR analysis to determine the expression levels of Tnfa, Il6, and Il10. Values are mean ± S.E. of four experiments. ctrl, control. B and C, naïve PMΦ (B) and PMΦ (C), classically primed with IFNγ, from wild-type and Nfkbiz−/− mice were either left untreated (ctrl) or cultured in the presence of 1 μg/ml LPS for 24 h before expression of Tnfa, Il6, and Il10 was analyzed by qRT-PCR. Values were normalized to Gapdh and are mean ± S.E. of four experiments. *, statistical significance comparing wild-type and Nfkbiz−/− cells; N.D., not detectable.
FIGURE 2.
FIGURE 2.
IκBζ deficiency reduces IL-10 secretion in bone marrow macrophages. A and B, naïve (A) and classically activated (B) wild-type and Nfkbiz−/− BMMΦ were cultured in the presence of 1 μg/ml LPS. Total RNA was subjected to qRT-PCR analysis to determine the expression levels of Tnfa, Il6, and Il10. Values are mean ± S.E. from five experiments. ctrl, control. C, wild-type and Nfkbiz−/− BMMΦ were cultured with 1 μg/ml LPS. Total RNA was isolated after the indicated time points and subjected to qRT-PCR analysis to determine the levels of Tnfa, Il6, and Il10 mRNA expression. Values are mean ± S.E. from four experiments. D–F, IL-10 (D), TNFα (E), and IL-6 (F) concentrations in cell culture supernatants were assessed at the indicated time points of LPS stimulation. Values are mean ± S.E. from three experiments. *, statistical significance comparing wild-type and Nfkbiz−/− cells; N.D., not detectable.
FIGURE 3.
FIGURE 3.
IκBζ-regulated IL-10 expression is independent of macrophage polarization. A–C, BMMΦ were either left naïve (A), classically activated with 30 ng/ml IFNγ (B), or alternatively activated with 20 ng/ml IL-4 (C) for 24 h before challenging or not with 1 μg/ml LPS for an additional 24 h. Culture supernatants were analyzed for the concentration of TNFα, IL-6 and IL-10. Values are mean ± S.E. of six experiments. *, statistical significance comparing luciferase activity in the presence and absence of doxycycline; N.D., not detectable. ctrl, control.
FIGURE 4.
FIGURE 4.
Ectopic expression of IκBζ induces Il10 expression. A, Raw264.7 cells were modified to enable the inducible doxycycline-dependent expression of IκBζ. Both Raw264.7 wild-type and the resulting Raw264.7/TetOn-IκBζ cells were cultured in the presence or absence of either 1 μg/ml LPS, 2 μg/ml doxycycline (Dox), or a combination thereof. After 24 h, induction of IκBζ expression was verified by immunoblot analysis. B, Raw264.7/TetOn-IκBζ cells were incubated in the presence of the indicated amounts of doxycycline. After 24 h, total RNA was isolated and subjected to qRT-PCR analysis for the expression of Il10, Il6, Nfkbiz, Nfkbia, and Elam1. Values are expressed as relative units (RU). Expression levels from cells treated with 2 μg/ml doxycycline were defined as 1 RU. Values are mean ± S.D. from three experiments. C, Raw264.7/TetOn-IκBζ cells were transfected with luciferase reporter gene constructs harboring the promoter region of the indicated genes or the empty vector control (ctrl). Cells were cultured in the presence of 2 μg/ml doxycycline for 48 h before luciferase assay was performed. Luciferase activity is given as percentage of the basal activity in the absence of doxycycline. Values are mean ± S.D. from three experiments. *, statistical significance comparing luciferase activity in the presence and absence of doxycycline.
FIGURE 5.
FIGURE 5.
IκBζ regulates Il10 promoter activity. A, schematic of the murine Il10 promoter. The dark boxes indicate the positions of κB binding sites. B, Raw264.7 cells were transfected with the empty luciferase reporter gene vector (ctrl) or reporter gene constructs harboring the indicated regions of the Il10 promoter. Luciferase activity was analyzed after 24 h of incubation in the absence or presence of LPS. Values are mean ± S.D. from three experiments. *, statistical significance comparing luciferase activity in the presence and absence of LPS. AU, arbitrary units. C, Raw264.7 cells were transfected with a luciferase reporter construct of a truncated Il10 promoter (−158 to +64 bp) harboring the proximal κB binding site together with pcDNA4 expression vectors for Nfkb1 (p50), IκBζ, or a combination thereof. Luciferase activity was analyzed 24 h after transfection. Promoter activity obtained after transfection of the empty pcDNA4 vector was set as 1. Values are mean ± S.D. of three experiments. D, chromatin from LPS-treated (5 h) naïve wild-type and Nfkbiz−/− BMMΦ was subjected to ChIP assays applying an H3K4me3-specific antibody. The degree of H3K4 trimethylation of the Il10, Il6, and Tnfa promoter was determined via qPCR. ChIP analysis with an isotype control antibody served as a control. Values are mean ± S.D. from three experiments. *, statistical significance comparing wild-type and Nfkbiz−/− cells.
FIGURE 6.
FIGURE 6.
IL-10 partially attenuates the M1-hyperpolarized state of Nfkbiz-deficient bone marrow macrophages. A, BMMΦ from wild-type and Nfkbiz−/− mice were cultured in the presence or absence of 1 μg/ml LPS for 5 h. Cells were subsequently assayed for the indicated mRNA levels. Values were normalized to Gapdh and are mean ± S.D. from four experiments. *, statistical significance comparing wild-type and knockout cells; N.D., not detectable. ctrl, control. B, wild type and Nfkbiz−/− BMMΦ were stimulated with 1 μg/ml LPS for the indicated times. The levels of Stat1, phospho-Stat1 (Tyr701), and β-actin were analyzed by immunoblotting. An exemplary set of data is shown. C and D, Nfkbiz−/− BMMΦ were cultured in the presence of 1 μg/ml LPS or left untreated. After 4 h, the supernatants were replaced by medium supplemented with the indicated concentrations of IL-10 (C) or by supernatants from wild-type BMMΦ (wt) challenged for 24 h with LPS (D). Samples for qRT-PCR were prepared after 4 h of incubation and analyzed for relative mRNA expression of Stat1 and Gbp4. Values are mean ± S.D. from three experiments.
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References

    1. Gordon S., and Taylor P. R. (2005) Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953–964 - PubMed
    1. Ginhoux F., and Jung S. (2014) Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat. Rev. Immunol 14, 392–404 - PubMed
    1. Laskin D. L., Sunil V. R., Gardner C. R., and Laskin J. D. (2011) Macrophages and tissue injury: agents of defense or destruction? Annu. Rev. Pharmacol. Toxicol. 51, 267–288 - PMC - PubMed
    1. Murray P. J., Allen J. E., Biswas S. K., Fisher E. A., Gilroy D. W., Goerdt S., Gordon S., Hamilton J. A., Ivashkiv L. B., Lawrence T., Locati M., Mantovani A., Martinez F. O., Mege J. L., Mosser D. M., et al. (2014) Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 41, 14–20 - PMC - PubMed
    1. Sica A., and Mantovani A. (2012) Macrophage plasticity and polarization: in vivo veritas. J. Clin. Invest. 122, 787–795 - PMC - PubMed

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