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

  • Sebastian Hörber
    Affiliations
    From the Interfaculty Institute of Biochemistry, Department of Molecular Medicine, University of Tübingen, 72076 Tübingen, Germany and
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  • Dominic G. Hildebrand
    Affiliations
    From the Interfaculty Institute of Biochemistry, Department of Molecular Medicine, University of Tübingen, 72076 Tübingen, Germany and
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  • Wolfgang S. Lieb
    Affiliations
    From the Interfaculty Institute of Biochemistry, Department of Molecular Medicine, University of Tübingen, 72076 Tübingen, Germany and
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  • Sebastian Lorscheid
    Affiliations
    From the Interfaculty Institute of Biochemistry, Department of Molecular Medicine, University of Tübingen, 72076 Tübingen, Germany and
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  • Stephan Hailfinger
    Affiliations
    From the Interfaculty Institute of Biochemistry, Department of Molecular Medicine, University of Tübingen, 72076 Tübingen, Germany and
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  • Klaus Schulze-Osthoff
    Correspondence
    To whom correspondence may be addressed: Interfaculty Institute of Biochemistry, Eberhard Karls University, 72076 Tübingen, Germany. Tel.: 49-7071-2973399; Fax: 49-7071-294017
    Affiliations
    From the Interfaculty Institute of Biochemistry, Department of Molecular Medicine, University of Tübingen, 72076 Tübingen, Germany and

    the German Cancer Consortium (DKTK) and German Cancer Research Center, 69120 Heidelberg, Germany
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  • Frank Essmann
    Correspondence
    To whom correspondence may be addressed: Interfaculty Institute of Biochemistry, Eberhard Karls University, 72076 Tübingen, Germany. Tel.: 49-7071-2974162; Fax: 49-7071-294017
    Affiliations
    From the Interfaculty Institute of Biochemistry, Department of Molecular Medicine, University of Tübingen, 72076 Tübingen, Germany and
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  • Author Footnotes
    * This work was supported by Deutsche Forschungsgemeinschaft Grants SFB685 and GRK1302 and Bundesminsterium für Bildung und Forschung Grant AID-NET. The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Figs. 1 and 2.
Open AccessPublished:April 18, 2016DOI:https://doi.org/10.1074/jbc.M116.718825
      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.

      Introduction

      Macrophages constitute a heterogeneous group of phagocytes that fulfill pro- as well as anti-inflammatory responses (
      • Gordon S.
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      Monocyte and macrophage heterogeneity.
      ,
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      Monocytes and macrophages: developmental pathways and tissue homeostasis.
      ). A pro-inflammatory response of innate immune cells is indispensable for host defense. However, dysregulation of innate immunity can result in severe damage of the affected host. Weak immune responses lead to prolonged infection and persistence of pathogens, whereas overshooting responses promote chronic inflammation and autoimmune disease (
      • Laskin D.L.
      • Sunil V.R.
      • Gardner C.R.
      • Laskin J.D.
      Macrophages and tissue injury: agents of defense or destruction?.
      ). Therefore, tight control of defense mechanisms is essential for host protection against self-destructive, excessive, and undue immune responses.
      For macrophages, two distinct states of polarization have been defined. Classically activated (M1-polarized) macrophages exert their pro-inflammatory role as effector cells in cell-mediated immune responses, whereas alternatively activated (M2-polarized) macrophages are involved in immunosuppression, wound healing, and tissue regeneration (
      • Murray P.J.
      • Allen J.E.
      • Biswas S.K.
      • Fisher E.A.
      • Gilroy D.W.
      • Goerdt S.
      • Gordon S.
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      • Mege J.L.
      • Mosser D.M.
      • et al.
      Macrophage activation and polarization: nomenclature and experimental guidelines.
      ). In contrast to M1 macrophages, the M2 counterparts secrete high amounts of anti-inflammatory cytokines, including the crucial immunosuppressive cytokine IL-10, thereby guaranteeing a balanced immune response (
      • Sica A.
      • Mantovani A.
      Macrophage plasticity and polarization: in vivo veritas.
      ).
      IL-10 can inhibit various macrophage functions, such as nitric oxide synthesis and pro-inflammatory cytokine production, as well as the expression of major histocompatibility complex proteins and co-stimulatory receptors (
      • Moore K.W.
      • de Waal Malefyt R.
      • Coffman R.L.
      • O'Garra A.
      Interleukin-10 and the interleukin-10 receptor.
      ,
      • Saraiva M.
      • O'Garra A.
      The regulation of IL-10 production by immune cells.
      ). In contrast, the absence of IL-10 results in spontaneous development of inflammatory bowel disease and increased pathological alterations caused by uncontrolled responses to infectious pathogens (
      • Kühn R.
      • Löhler J.
      • Rennick D.
      • Rajewsky K.
      • Müller W.
      Interleukin-10-deficient mice develop chronic enterocolitis.
      ,
      • O'Garra A.
      • Vieira P.L.
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      • Goldfeld A.E.
      IL-10-producing and naturally occurring CD4+ Tregs: limiting collateral damage.
      ). IL-10 may also act as a negative feedback regulator of chronic infectious diseases by inhibiting IL-6, IL-12, and TNFα secretion, thereby keeping immune responses in check and preventing tissue damage (
      • O'Garra A.
      • Vieira P.L.
      • Vieira P.
      • Goldfeld A.E.
      IL-10-producing and naturally occurring CD4+ Tregs: limiting collateral damage.
      ). Furthermore, the administration of exogenous IL-10 has been shown to ameliorate inflammatory and autoimmune diseases in several animal models (
      • Kühn R.
      • Löhler J.
      • Rennick D.
      • Rajewsky K.
      • Müller W.
      Interleukin-10-deficient mice develop chronic enterocolitis.
      ).
      An important regulator for the expression of cytokines and other immune regulators is the transcription factor NF-κB. The NF-κB family consists of five members that bind as homo- or heterodimers at κB sites in the DNA of target genes (
      • Oeckinghaus A.
      • Hayden M.S.
      • Ghosh S.
      Crosstalk in NF-κB signaling pathways.
      ). Depending on their transactivation activity, the NF-κB subunits can be divided into two subgroups. RelA (p65), RelB, and c-Rel possess a C-terminal transcription activation domain, whereas p50 (Nfkb1) and p52 (Nfkb2) lack a transcription activation domain. Based on these structural differences, NF-κB dimers containing at least one subunit with a transcription activation domain act as transcriptional activators, whereas p50/p50 or p52/p52 homodimers are assumed to function as transcriptional repressors.
      Because various stimuli activate the NF-κB signaling pathway and a great diversity of target genes is regulated by NF-κB, a precise control of NF-κB activity is required to avoid misguided immune responses. In fact, NF-κB activation is controlled by a series of cytosolic and nuclear regulatory events, in which IκB proteins play a pivotal role (
      • Oeckinghaus A.
      • Hayden M.S.
      • Ghosh S.
      Crosstalk in NF-κB signaling pathways.
      ,
      • Hinz M.
      • Arslan S.Ç.
      • Scheidereit C.
      It takes two to tango: IκBs, the multifunctional partners of NF-κB.
      ). In unstimulated cells, NF-κB is sequestered as an inactive complex bound to cytosolic IκB proteins such as IκBα, IκBβ, and IκB∈. Various stimuli cause the phosphorylation of cytosolic IκBs, leading to their proteasomal degradation, which subsequently enables NF-κB to translocate to the nucleus and activate target genes.
      Despite the presence of high-affinity binding sites, only a fraction of NF-κB target genes is generally activated in response to an inflammatory stimulus. It was suggested that NF-κB target genes can be categorized in two groups based on their kinetics of induction and the requirement of protein synthesis (
      • Smale S.T.
      Hierarchies of NF-κB target-gene regulation.
      ,
      • Smale S.T.
      Selective transcription in response to an inflammatory stimulus.
      ). Although primary NF-κB response genes are rapidly induced, the expression of secondary target genes is delayed and requires the prior synthesis of additional NF-κB coregulators. A novel and emerging group of such NF-κB coregulators are so-called atypical IκB proteins, including Bcl-3, IκBNS, IκBζ, and IκBβ (
      • Hinz M.
      • Arslan S.Ç.
      • Scheidereit C.
      It takes two to tango: IκBs, the multifunctional partners of NF-κB.
      ,
      • Schuster M.
      • Annemann M.
      • Plaza-Sirvent C.
      • Schmitz I.
      Atypical IκB proteins: nuclear modulators of NF-κB signaling.
      ). Atypical IκBs differ markedly from classical cytosolic IκBs because they are mostly inducibly expressed and localized in the nucleus. Moreover, atypical IκBs do not exclusively act as inhibitors but can also activate the expression of secondary response genes.
      The atypical IκB protein IκBζ has been recently implicated in differential NF-κB target gene expression in macrophages (
      • Hildebrand D.G.
      • Alexander E.
      • Hörber S.
      • Lehle S.
      • Obermayer K.
      • Münck N.A.
      • Rothfuss O.
      • Frick J.S.
      • Morimatsu M.
      • Schmitz I.
      • Roth J.
      • Ehrchen J.M.
      • Essmann F.
      • Schulze-Osthoff K.
      IκBζ is a transcriptional key regulator of CCL2/MCP-1.
      ,
      • Yamamoto M.
      • Yamazaki S.
      • Uematsu S.
      • Sato S.
      • Hemmi H.
      • Hoshino K.
      • Kaisho T.
      • Kuwata H.
      • Takeuchi O.
      • Takeshige K.
      • Saitoh T.
      • Yamaoka S.
      • Yamamoto N.
      • Yamamoto S.
      • Muta T.
      • et al.
      Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ.
      ) even though its physiological function remains largely unknown. The IκBζ-encoding Nfkbiz gene is rapidly induced as a primary NF-κB response gene by various inflammatory stimuli and, through association with the NF-κB subunit p50, is thought to exert its transcription-enhancing activity on secondary response genes mainly at the level of chromatin remodeling (
      • Smale S.T.
      Hierarchies of NF-κB target-gene regulation.
      ,
      • Totzke G.
      • Essmann F.
      • Pohlmann S.
      • Lindenblatt C.
      • Jänicke R.U.
      • Schulze-Osthoff K.
      A novel member of the IκB family, human IκB-ζ, inhibits transactivation of p65 and its DNA binding.
      ,
      • Trinh D.V.
      • Zhu N.
      • Farhang G.
      • Kim B.J.
      • Huxford T.
      The nuclear IκB protein IκBζ specifically binds NF-κB p50 homodimers and forms a ternary complex on κB DNA.
      ).
      Recently, we showed that in macrophages, expression of CCL2 strictly depends on the presence of IκBζ and, consequently, that expression of CCL2 is abolished in macrophages from IκBζ-deficient mice (
      • Hildebrand D.G.
      • Alexander E.
      • Hörber S.
      • Lehle S.
      • Obermayer K.
      • Münck N.A.
      • Rothfuss O.
      • Frick J.S.
      • Morimatsu M.
      • Schmitz I.
      • Roth J.
      • Ehrchen J.M.
      • Essmann F.
      • Schulze-Osthoff K.
      IκBζ is a transcriptional key regulator of CCL2/MCP-1.
      ). Furthermore, several other pro-inflammatory gene products, including IL-6, IL12p40, IL-17, IFNγ, and GM-CSF, have been found to be regulated by IκBζ (
      • Yamamoto M.
      • Yamazaki S.
      • Uematsu S.
      • Sato S.
      • Hemmi H.
      • Hoshino K.
      • Kaisho T.
      • Kuwata H.
      • Takeuchi O.
      • Takeshige K.
      • Saitoh T.
      • Yamaoka S.
      • Yamamoto N.
      • Yamamoto S.
      • Muta T.
      • et al.
      Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ.
      ,
      • Kayama H.
      • Ramirez-Carrozzi V.R.
      • Yamamoto M.
      • Mizutani T.
      • Kuwata H.
      • Iba H.
      • Matsumoto M.
      • Honda K.
      • Smale S.T.
      • Takeda K.
      Class-specific regulation of pro-inflammatory genes by MyD88 pathways and IκBζ.
      ,
      • Okamoto K.
      • Iwai Y.
      • Oh-Hora M.
      • Yamamoto M.
      • Morio T.
      • Aoki K.
      • Ohya K.
      • Jetten A.M.
      • Akira S.
      • Muta T.
      • Takayanagi H.
      IκBζ regulates T(H)17 development by cooperating with ROR nuclear receptors.
      ,
      • Kannan Y.
      • Yu J.
      • Raices R.M.
      • Seshadri S.
      • Wei M.
      • Caligiuri M.A.
      • Wewers M.D.
      IκBζ augments IL-12- and IL-18-mediated IFN-γ production in human NK cells.
      ,
      • Alexander E.
      • Hildebrand D.G.
      • Kriebs A.
      • Obermayer K.
      • Manz M.
      • Rothfuss O.
      • Schulze-Osthoff K.
      • Essmann F.
      IκBζ is a regulator of the senescence-associated secretory phenotype in DNA damage- and oncogene-induced senescence.
      ,
      • Johansen C.
      • Mose M.
      • Ommen P.
      • Bertelsen T.
      • Vinter H.
      • Hailfinger S.
      • Lorscheid S.
      • Schulze-Osthoff K.
      • Iversen L.
      IκBζ is a key driver in the development of psoriasis.
      ). Intriguingly, however, despite impaired expression of these pro-inflammatory cytokines, Nfkbiz−/− mice display a pro-inflammatory phenotype characterized by periocular inflammation, inflammatory skin alterations, and an M1 hyperpolarized macrophage state (
      • Hildebrand D.G.
      • Alexander E.
      • Hörber S.
      • Lehle S.
      • Obermayer K.
      • Münck N.A.
      • Rothfuss O.
      • Frick J.S.
      • Morimatsu M.
      • Schmitz I.
      • Roth J.
      • Ehrchen J.M.
      • Essmann F.
      • Schulze-Osthoff K.
      IκBζ is a transcriptional key regulator of CCL2/MCP-1.
      ,
      • Yamamoto M.
      • Yamazaki S.
      • Uematsu S.
      • Sato S.
      • Hemmi H.
      • Hoshino K.
      • Kaisho T.
      • Kuwata H.
      • Takeuchi O.
      • Takeshige K.
      • Saitoh T.
      • Yamaoka S.
      • Yamamoto N.
      • Yamamoto S.
      • Muta T.
      • et al.
      Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ.
      ,
      • Shiina T.
      • Konno A.
      • Oonuma T.
      • Kitamura H.
      • Imaoka K.
      • Takeda N.
      • Todokoro K.
      • Morimatsu M.
      Targeted disruption of MAIL, a nuclear IκB protein, leads to severe atopic dermatitis-like disease.
      ,
      • Okuma A.
      • Hoshino K.
      • Ohba T.
      • Fukushi S.
      • Aiba S.
      • Akira S.
      • Ono M.
      • Kaisho T.
      • Muta T.
      Enhanced apoptosis by disruption of the STAT3-IκB-ζ signaling pathway in epithelial cells induces Sjögren's syndrome-like autoimmune disease.
      ). In view of the phenotype of Nfkbiz−/− mice, we therefore investigated a potential role of IκBζ for the regulation of IL-10 as an essential anti-inflammatory cytokine. Interestingly, although expression of Il10 has been found previously to be inhibited by the atypical IκB protein Bcl-3 (
      • Kuwata H.
      • Watanabe Y.
      • Miyoshi H.
      • Yamamoto M.
      • Kaisho T.
      • Takeda K.
      • Akira S.
      IL-10-inducible Bcl-3 negatively regulates LPS-induced TNF-α production in macrophages.
      ,
      • Riemann M.
      • Endres R.
      • Liptay S.
      • Pfeffer K.
      • Schmid R.M.
      The IκB protein Bcl-3 negatively regulates transcription of the IL-10 gene in macrophages.
      ), our results establish IκBζ as a novel and essential transcriptional inducer of Il10 in macrophages. Our results therefore show for the first time that IκBζ is not only a pro-inflammatory mediator but also controls the activation of anti-inflammatory gene products.

      Discussion

      Growing evidence suggests that the induction of NF-κB-regulated genes is not solely defined by the nuclear translocation of NF-κB but that different NF-κB target genes have individual expression profiles regarding kinetic, stimulus, or cell type, thereby ensuring a selectivity of an immune response. Several recent studies identified a subfamily of atypical IκB proteins as important “specifiers” that select particular κB-sites to be activated or repressed under certain conditions (
      • Schuster M.
      • Annemann M.
      • Plaza-Sirvent C.
      • Schmitz I.
      Atypical IκB proteins: nuclear modulators of NF-κB signaling.
      ).
      IκBζ is mostly regarded as a pro-inflammatory regulator, as demonstrated e.g. by its requirement for Th17 differentiation and expression of particular pro-inflammatory cytokines. Nevertheless, Nfkbiz−/− mice show a pro-inflammatory phenotype and M1 hyperpolarization of macrophages (
      • Hildebrand D.G.
      • Alexander E.
      • Hörber S.
      • Lehle S.
      • Obermayer K.
      • Münck N.A.
      • Rothfuss O.
      • Frick J.S.
      • Morimatsu M.
      • Schmitz I.
      • Roth J.
      • Ehrchen J.M.
      • Essmann F.
      • Schulze-Osthoff K.
      IκBζ is a transcriptional key regulator of CCL2/MCP-1.
      ,
      • Yamamoto M.
      • Yamazaki S.
      • Uematsu S.
      • Sato S.
      • Hemmi H.
      • Hoshino K.
      • Kaisho T.
      • Kuwata H.
      • Takeuchi O.
      • Takeshige K.
      • Saitoh T.
      • Yamaoka S.
      • Yamamoto N.
      • Yamamoto S.
      • Muta T.
      • et al.
      Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ.
      ,
      • Shiina T.
      • Konno A.
      • Oonuma T.
      • Kitamura H.
      • Imaoka K.
      • Takeda N.
      • Todokoro K.
      • Morimatsu M.
      Targeted disruption of MAIL, a nuclear IκB protein, leads to severe atopic dermatitis-like disease.
      ,
      • Okuma A.
      • Hoshino K.
      • Ohba T.
      • Fukushi S.
      • Aiba S.
      • Akira S.
      • Ono M.
      • Kaisho T.
      • Muta T.
      Enhanced apoptosis by disruption of the STAT3-IκB-ζ signaling pathway in epithelial cells induces Sjögren's syndrome-like autoimmune disease.
      ), suggesting that so far unknown anti-inflammatory mediators might be controlled by IκBζ. In this study, we found that induction of the potent anti-inflammatory cytokine IL-10 by LPS but also by TLR2 agonists (data not shown) was strictly dependent on IκBζ and strongly reduced in Nfkbiz−/− mice. These results were supported by the finding that the doxycycline-inducible expression of ectopic IκBζ in Raw264.7 macrophages tightly correlated with increasing Il10 mRNA levels. Moreover, reporter analysis revealed that the proximal κB site of the Il10 promoter was responsible for IκBζ-mediated Il10 expression. The recruitment of IκBζ was associated with histone H3K4 trimethylation of the proximal promoter region as a marker of active gene transcription. Interestingly, in the absence of IκBζ, H3K4 trimethylation did not occur, which, along with other lines of evidence, suggests that chromatin remodeling is essential for IκBζ action. Thus, our results in knockout and overexpression models suggest that transcriptional regulation of Il10 directly depends on IκBζ.
      So far, the expression of IL-10 in macrophages is known to be primarily regulated by transcription factors such as SP1, C/EBPβ, IRF1, and STAT3 (
      • Brightbill H.D.
      • Plevy S.E.
      • Modlin R.L.
      • Smale S.T.
      A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages.
      ,
      • Tone M.
      • Powell M.J.
      • Tone Y.
      • Thompson S.A.
      • Waldmann H.
      IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3.
      ,
      • Brenner S.
      • Prösch S.
      • Schenke-Layland K.
      • Riese U.
      • Gausmann U.
      • Platzer C.
      cAMP-induced interleukin-10 promoter activation depends on CCAAT/enhancer-binding protein expression and monocytic differentiation.
      ,
      • Ziegler-Heitbrock L.
      • Lötzerich M.
      • Schaefer A.
      • Werner T.
      • Frankenberger M.
      • Benkhart E.
      IFN-α induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3.
      ), whereas a role of different NF-κB proteins is relatively unknown. Because of the lack of a transcription activation domain, p50 NF-κB homodimers, which retain their ability to bind to κB sites, are thought to be transcriptional repressors. Interestingly, although not investigating atypical IκB proteins, earlier studies already showed that p50 homodimers bind to the proximal Il10 promoter and activate Il10 transcription in primary macrophages (
      • Cao S.
      • Zhang X.
      • Edwards J.P.
      • Mosser D.M.
      NF-κB1 (p50) homodimers differentially regulate pro- and anti-inflammatory cytokines in macrophages.
      ).
      Moreover, in contrast to IκBζ, Bcl-3, a related atypical IκB protein that also requires p50 for co-regulation, negatively regulates Il10 transcription in macrophages (
      • Riemann M.
      • Endres R.
      • Liptay S.
      • Pfeffer K.
      • Schmid R.M.
      The IκB protein Bcl-3 negatively regulates transcription of the IL-10 gene in macrophages.
      ,
      • Wessells J.
      • Baer M.
      • Young H.A.
      • Claudio E.
      • Brown K.
      • Siebenlist U.
      • Johnson P.F.
      BCL-3 and NF-κB p50 attenuate lipopolysaccharide-induced inflammatory responses in macrophages.
      ), although the exact role of Bcl-3 for Il10 expression is controversial (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). Bcl3 knockout mice show enhanced susceptibility to infection with Listeria monocytogenes, which is due to enhanced expression of IL-10, resulting in diminished levels of IL-12p70 and IFNγ. These results suggest that atypical IκB proteins, such as Bcl-3 and IκBζ, might regulate gene expression in an opposite manner, which is also underlined by the fact that IL-12p70 and IFNγ are direct IκBζ targets. Likewise, Bcl-3 and IκBζ have an antagonistic effect on CCL2 expression in macrophages. Although Bcl-3 inhibits the expression of CCL2, IκBζ promotes the expression of this chemokine (
      • Hildebrand D.G.
      • Alexander E.
      • Hörber S.
      • Lehle S.
      • Obermayer K.
      • Münck N.A.
      • Rothfuss O.
      • Frick J.S.
      • Morimatsu M.
      • Schmitz I.
      • Roth J.
      • Ehrchen J.M.
      • Essmann F.
      • Schulze-Osthoff K.
      IκBζ is a transcriptional key regulator of CCL2/MCP-1.
      ,
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). Another example of such opposite gene regulation by atypical IκB proteins concerns IκBζ and IκBNS. For instance, although IκBζ is required for IL-6, IL-12p40, and G-CSF expression (
      • Hildebrand D.G.
      • Alexander E.
      • Hörber S.
      • Lehle S.
      • Obermayer K.
      • Münck N.A.
      • Rothfuss O.
      • Frick J.S.
      • Morimatsu M.
      • Schmitz I.
      • Roth J.
      • Ehrchen J.M.
      • Essmann F.
      • Schulze-Osthoff K.
      IκBζ is a transcriptional key regulator of CCL2/MCP-1.
      ,
      • Yamamoto M.
      • Yamazaki S.
      • Uematsu S.
      • Sato S.
      • Hemmi H.
      • Hoshino K.
      • Kaisho T.
      • Kuwata H.
      • Takeuchi O.
      • Takeshige K.
      • Saitoh T.
      • Yamaoka S.
      • Yamamoto N.
      • Yamamoto S.
      • Muta T.
      • et al.
      Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ.
      ), IκBNS apparently inhibits transcription of these cytokines (
      • Hirotani T.
      • Lee P.Y.
      • Kuwata H.
      • Yamamoto M.
      • Matsumoto M.
      • Kawase I.
      • Akira S.
      • Takeda K.
      The nuclear IκB protein IκBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria.
      ,
      • Kuwata H.
      • Matsumoto M.
      • Atarashi K.
      • Morishita H.
      • Hirotani T.
      • Koga R.
      • Takeda K.
      IκBNS inhibits induction of a subset of Toll-like receptor-dependent genes and limits inflammation.
      ). Interestingly, our gene expression profiling suggest that atypical IκB proteins might also influence each other at the transcriptional level and, moreover, compete with each other for p50-mediated DNA binding. Together, these findings suggest that atypical IκB proteins form a complex network in controlling NF-κB responses.
      In addition to transcription factor binding, previous studies suggested that Il10 expression is regulated by changes in the chromatin structure at the IL10 locus. The histone deacetylase HDAC11 has been found to inhibit IL-10 expression (
      • Villagra A.
      • Cheng F.
      • Wang H.W.
      • Suarez I.
      • Glozak M.
      • Maurin M.
      • Nguyen D.
      • Wright K.L.
      • Atadja P.W.
      • Bhalla K.
      • Pinilla-Ibarz J.
      • Seto E.
      • Sotomayor E.M.
      The histone deacetylase HDAC11 regulates the expression of interleukin 10 and immune tolerance.
      ), whereas phosphorylation of histone H3 at serine 10 is needed for transcriptional activation of the Il10 promoter (
      • Lucas M.
      • Zhang X.
      • Prasanna V.
      • Mosser D.M.
      ERK activation following macrophage FcγR ligation leads to chromatin modifications at the IL-10 locus.
      ). A recent study found that IκBζ recruits the epigenetic modifier Tet2 to selective promoter regions independent of DNA methylation (
      • Zhang Q.
      • Zhao K.
      • Shen Q.
      • Han Y.
      • Gu Y.
      • Li X.
      • Zhao D.
      • Liu Y.
      • Wang C.
      • Zhang X.
      • Su X.
      • Liu J.
      • Ge W.
      • Levine R.L.
      • Li N.
      • Cao X.
      Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6.
      ). IκBζ further mediates chromatin remodeling by recruiting the SWI/SNF complex to target genes, thereby enhancing promoter accessibility (
      • Tartey S.
      • Matsushita K.
      • Vandenbon A.
      • Ori D.
      • Imamura T.
      • Mino T.
      • Standley D.M.
      • Hoffmann J.A.
      • Reichhart J.M.
      • Akira S.
      • Takeuchi O.
      Akirin2 is critical for inducing inflammatory genes by bridging IκB-ζ and the SWI/SNF complex.
      ). The same mechanism presumably underlies the regulation of Il10 expression because we found that Il10 promoter accessibility and H3K4 trimethylation were reduced in Nfkbiz−/− cells. Thus, it will be interesting to explore whether Bcl-3 and IκBζ mediate their antagonistic effects at the Il10 promoter by recruiting distinct histone-modifying enzymes.
      Although cells of the macrophage lineage are a major source of IL-10, several other cell types of the innate and adaptive immune system can express this cytokine (
      • Moore K.W.
      • de Waal Malefyt R.
      • Coffman R.L.
      • O'Garra A.
      Interleukin-10 and the interleukin-10 receptor.
      ,
      • Saraiva M.
      • O'Garra A.
      The regulation of IL-10 production by immune cells.
      ). Further studies are needed to explore whether the strict control of IL-10 expression by IκBζ is also relevant to other cell types. Our exemplary investigation of wild-type and Nfkbiz−/− MEFs indicates that the described mechanism is not restricted to macrophages. Interestingly, previous gene expression analysis revealed that IκBζ-deficient macrophages show a bias toward M1 polarization, evidenced by the increased expression of certain M1 markers (
      • Hildebrand D.G.
      • Alexander E.
      • Hörber S.
      • Lehle S.
      • Obermayer K.
      • Münck N.A.
      • Rothfuss O.
      • Frick J.S.
      • Morimatsu M.
      • Schmitz I.
      • Roth J.
      • Ehrchen J.M.
      • Essmann F.
      • Schulze-Osthoff K.
      IκBζ is a transcriptional key regulator of CCL2/MCP-1.
      ). It is worth mentioning that p50-deficient mice also show exacerbated M1-driven inflammation and reduced M2 polarization of their macrophages (
      • Porta C.
      • Rimoldi M.
      • Raes G.
      • Brys L.
      • Ghezzi P.
      • Di Liberto D.
      • Dieli F.
      • Ghisletti S.
      • Natoli G.
      • De Baetselier P.
      • Mantovani A.
      • Sica A.
      Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor κB.
      ), although several phenotypic alterations are distinct between Nfkbiz−/− and p50-deficient mice.
      In functional studies, we found that the increased mRNA expression of Stat1 and Gpb4 could be partially reverted not only by IL-10 supplementation but also by IL-10-proficient supernatants from wild-type macrophages even though no reduction in Cxcl9 expression was observed. It was not the intention of our study to investigate the role of IκBζ-mediated IL-10 expression in macrophage polarization. Our results, however, indicate that decreased IL-10 expression contributes to at least some of the features of M1 polarization in Nfkbiz−/− mice. In line, IL-10-producing monocytes have been found to preferentially differentiate to M2 macrophages (
      • Prasse A.
      • Germann M.
      • Pechkovsky D.V.
      • Markert A.
      • Verres T.
      • Stahl M.
      • Melchers I.
      • Luttmann W.
      • Müller-Quernheim J.
      • Zissel G.
      IL-10-producing monocytes differentiate to alternatively activated macrophages and are increased in atopic patients.
      ,
      • Makita N.
      • Hizukuri Y.
      • Yamashiro K.
      • Murakawa M.
      • Hayashi Y.
      IL-10 enhances the phenotype of M2 macrophages induced by IL-4 and confers the ability to increase eosinophil migration.
      ).
      Dysregulation of Il10 expression has been linked to several immune disorders. Transgenic mice overexpressing IL-10 in macrophages exhibit increased susceptibility to bacterial infections and septic shock (
      • Lang R.
      • Rutschman R.L.
      • Greaves D.R.
      • Murray P.J.
      Autocrine deactivation of macrophages in transgenic mice constitutively overexpressing IL-10 under control of the human CD68 promoter.
      ). Excessive IL-10 secretion has also been linked to impaired tumor immune surveillance (
      • Béguelin W.
      • Sawh S.
      • Chambwe N.
      • Chan F.C.
      • Jiang Y.
      • Choo J.W.
      • Scott D.W.
      • Chalmers A.
      • Geng H.
      • Tsikitas L.
      • Tam W.
      • Bhagat G.
      • Gascoyne R.D.
      • Shaknovich R.
      IL10 receptor is a novel therapeutic target in DLBCLs.
      ,
      • Ruffell B.
      • Chang-Strachan D.
      • Chan V.
      • Rosenbusch A.
      • Ho C.M.
      • Pryer N.
      • Daniel D.
      • Hwang E.S.
      • Rugo H.S.
      • Coussens L.M.
      Macrophage IL-10 blocks CD8+ T cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells.
      ). In contrast, the absence of IL-10 results in spontaneous inflammatory bowel disease (
      • Kühn R.
      • Löhler J.
      • Rennick D.
      • Rajewsky K.
      • Müller W.
      Interleukin-10-deficient mice develop chronic enterocolitis.
      ), emphasizing its protective role in inflammatory and autoimmune conditions. We did not detect spontaneous colitis in Nfkbiz−/− mice, which might be caused by the genetic background because intestinal lesions have been reported to be least severe in C57BL/6 mice (
      • Berg D.J.
      • Davidson N.
      • Kühn R.
      • Müller W.
      • Menon S.
      • Holland G.
      • Thompson-Snipes L.
      • Leach M.W.
      • Rennick D.
      Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4 TH1-like responses.
      ). It is, however, worth mentioning that Nfkbiz−/− mice exhibit an increased susceptibility to dextran sodium sulfate-induced colitis.
      S. Hörber, D. G. Hildebrand, S. Lorscheid, J. S. Frick, K. Schulze-Osthoff, and F. Essmann, unpublished results.
      In summary, we have uncovered an essential novel regulatory mechanism of Il10 gene regulation in macrophages. We demonstrate that IκBζ through p50-mediated recruitment to the proximal Il10 promoter and subsequent histone H3 modification, enables transcription of the Il10 locus. Because IL-10 plays a beneficial role in several inflammatory diseases, Nfkbiz−/− mice are an interesting model system for evaluating IκBζ as a potential therapeutic target in inflammatory diseases.

      Author Contributions

      Se. H., D. G. H., K. S. O., and F. E. designed the research. Se. H., D. G. H., W. S. L., and S. L. performed the research. Se. H., D. G. H., W. S. L., and F. E. analyzed the data. St. H., K. S. O., and F. E. wrote the paper.

      Acknowledgments

      We thank S. Gaffen, M. Morimatsu, S. Smale, I. Schmitz, and G. Totzke for reagents.

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