NF-κB1 Inhibits TLR-Induced IFN-β Production in Macrophages through TPL-2− Dependent ERK Activation

This information is current as Huei-Ting Yang, Yanyan Wang, Xixing Zhao, Ezana of October 1, 2021. Demissie, Stamatia Papoutsopoulou, Agnes Mambole, Anne O'Garra, Michal F. Tomczak, Susan E. Erdman, James G. Fox, Steven C. Ley and Bruce H. Horwitz J Immunol published online 7 January 2011 http://www.jimmunol.org/content/early/2011/01/07/jimmun ol.1001003 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2011/01/07/jimmunol.100100

Material 3.DC1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 7, 2011, doi:10.4049/jimmunol.1001003 The Journal of Immunology

NF-kB1 Inhibits TLR-Induced IFN-b Production in Macrophages through TPL-2–Dependent ERK Activation

Huei-Ting Yang,*,1 Yanyan Wang,†,1 Xixing Zhao,† Ezana Demissie,† Stamatia Papoutsopoulou,* Agnes Mambole,* Anne O’Garra,‡ Michal F. Tomczak,† Susan E. Erdman,x James G. Fox,x Steven C. Ley,* and Bruce H. Horwitz†,{

Although NF-kB1 p50/p105 has critical roles in immunity, the mechanism by which NF-kB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb12/2 macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-b expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-b production. Markedly b 2/2 higher serum levels of IFN- were observed in Nfkb1 mice than in wild-type mice following LPS injection, demonstrating that Downloaded from Nfkb1 inhibits IFN-b production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabi- lized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-b production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb12/2 macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-b secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb12/2 macrophages, which rescued LPS activation of ERK, also inhibited IFN-b expression. These data indicate that p50/

p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of http://www.jimmunol.org/ ERK. The Journal of Immunology, 2011, 186: 000–000.

he NF-kB family of transcription factors, which share an promoter and enhancer regions of many genes involved in immune N-terminal Rel-homology domain, includes five subunits, and inflammatory responses (1, 4). p65, c-Rel, and RelB, which T NF-kB1 p50, p65 (RelA), c-Rel, RelB, and NF-kB2 p52 each have a C-terminal trans-activation domain, are synthesized in (1). These proteins form homo- and heterodimers that are retained their mature form. In contrast, p50 and p52 are produced from the N in the cytoplasm of unstimulated cells by members of the IkB termini of larger precursor proteins NF-kB1 p105 and NF-kB2 family of ankyrin repeat-containing proteins (IkB-a,-b, and -ε) p100, respectively, by proteolytic removal of their C termini by the by guest on October 1, 2021 (2). Inflammatory stimuli activate the IkB kinase complex (IKK), proteasome, and lack trans-activation domains (2). which phosphorylates two serines found within the N termini of NF-kB1 p50 induces transcription in heterodimeric complexes the IkBs, directing proteosomal degradation (3). IkB degradation with trans-activating Rel subunits. However, p50 homodimers can allows nuclear translocation of liberated NF-kB dimers, which then repress transcription by interfering with the binding of other active modulate transcription by binding to kB sites found within the NF-kB dimers and recruiting histone deacetylases to the promo- ter regions of kB-target genes (5, 6). It has been suggested that *Division of Immune Cell Biology, National Institute for Medical Research, London, p50 homodimers are responsible for inhibiting TNF expression United Kingdom; ‡Division of Immunoregulation, National Institute for Medical in LPS-tolerized macrophages (7–9), and maintaining an anti- Research, London, United Kingdom; †Department of Pathology, Brigham and Wom- x inflammatory phenotype in M2-polarized tumor-associated mac- en’s Hospital, Boston, MA 02115; Division of Comparative Medicine, Cambridge, MA 02139; and {Division of Emergency Medicine, Children’s Hospital, Boston, MA rophages (9, 10). p105 also has NF-kB inhibitory activity, due to 02115 its C-terminal ankyrin repeats. These are homologous to those 1H.-T.Y. and Y.W. contributed equally to this work. found in the classical IkB proteins, and retain NF-kB dimers in Received for publication March 29, 2010. Accepted for publication December 8, the cytoplasm of unstimulated cells. IKK phosphorylation of two 2010. serines in the C-terminal region of p105 after agonist stimulation This work was supported by National Institutes of Health Grants AI52267 (to B.H.H. leads to proteosomal-dependent complete degradation of p105 and S.E.E.), CA108854 (to S.E.E.), and CA67529 (to J.G.F.). H.-T.Y., S.P., and S.C.L. were supported by the United Kingdom Medical Research Council. (11–13), releasing associated p50, p65, and c-Rel to translocate into the nucleus (14). The C-terminal portion of p105 additionally The microarray data presented in this article have been submitted to the National Center for Biotechnology Information’s Gene Expression Omnibus under accession binds to TPL-2 (also known as MAP3K8 and Cot) (11, 15), a number GSE19941. mitogen-activated protein kinase kinase kinase that is essential Address correspondence and reprint requests to Dr. Bruce H. Horwitz or Dr. Steven for ERK activation following LPS stimulation of macrophages C. Ley, Department of Pathology, Brigham and Women’s Hospital, HNRB 630E, (16). Binding of p105 prevents TPL-2 from phosphorylating its 77 Avenue Louis Pasteur, Boston, MA 02115 (B.H.H.), or Division of Immune Cell Biology, National Institute for Medical Research, Mill Hill, London NW7 downstream targets, MEK-1/2, and is also required to maintain 1AA, United Kingdom (S.C.L.). E-mail addresses: [email protected]. TPL-2 protein stability. Consequently, LPS-induced activation edu (B.H.H.) and [email protected] (S.C.L.) of ERK-1/2 is blocked in bone marrow-derived macrophages The online version of this article contains supplemental material. (BMDMs) that lack p50/p105, due to substantially decreased Abbreviations used in this article: BMDM, bone marrow-derived macrophage; IKK, amounts of TPL-2 protein (17). IkB kinase complex; WT, wild-type. The multiple biochemical functions of NF-kB1 p50/p105 sug- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 gest that its role in immune responses is complex. This is consistent

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001003 2 NF-kB1 INHIBITS TLR-INDUCED IFN SIGNALING with observations that mice lacking p50/p105 exhibit both multi- normalization, and summarization of the probe-level data into probe set focal immune defects and increased sensitivity to inflammation (18, expression values were accomplished using Robust Multi-Array Analysis. 19). To further define the function of p50/p105 during TLR-induced Differential expression between samples was analyzed using the LIMMA package of the afflylmGUI within the Bioconductor R environment (22). The inflammatory responses, we profiled gene expression following 2 2 microarray data discussed in this publication are accessible through National LPS stimulation of Nfkb1 / macrophages. This analysis revealed Center for Biotechnology Information’s Gene Expression Omnibus, ac- that macrophages lacking p50/p105 demonstrated augmented in- cession number GSE19941 (http://www.ncbi.nlm.nih.gov/geo). duction of a type I IFN response. We show that the ability of NF- RT-PCR kB1 p50/p105 to inhibit this response depends on its ability to stabilize TPL-2 and facilitate TLR-induced ERK activation. Thus, RT-PCR analysis was performed using probes from Applied Biosystems (Foster City, CA), as per the manufacturer’s instructions. Expression for p50/p105-dependent ERK activation plays an important role in each sample was analyzed in duplicate and normalized between samples regulating the nature of TLR-induced inflammatory responses. using the DD cycle threshold method. Fold-change is reported as relative to uninduced levels in control BMDM. Each data point represents the average 6 SEM for two or three independent macrophage pools. Experiments are Materials and Methods representative of two or three independent experiments with consistent Experimental animals results. Il102/2, Nfkb12/2Il102/2, and Il102/2Rag22/2 mice were maintained on ELISA the 129S6/SvEvTac background, as previously described (20, 21). Nfkb12/2 2 2 2 2 2 2 2 2 IFN-b was analyzed in culture supernatants and serum using a commer- Il10 / Rag / mice were generated by crossing Nfkb1 / Il10 / and 2 2 2 2 2 2 cially available IFN-b–specific ELISA kit (PBL Biomedical Laboratories), Il10 / Rag2 / mice. Map3k8 / mice (provided by Professor Philip according to the manufacturer’s instructions.

Tsichlis, Tufts University, Boston and Thomas Jefferson University, Phil- Downloaded from adelphia, PA) were on the C57BL/6 background. In experiments compar- Immunoblotting ing Map3k82/2 mice with wild-type (WT) and Nfkb12/2 mice, all strains were on the C57BL/6 background. ERK (p44/42 MAPK), phospho-ERK (p44/42 MAPK) (Thr202/Tyr204), and phospho-STAT1 (Y701) Abs were purchased from Cell Signaling Tech- In vitro stimulation of BMDM nology (Beverly, MA). STAT1 p84/p91 (E-23), TFIID (Sl-1), c-Fos (sc- 52), and TPL-2 (sc-726) Abs were purchased from Santa Cruz Bio- BMDM were grown as previously described (20) and stimulated with LPS technology (Santa Cruz, CA).

from Escherichia coli 0127:B8 (Sigma-Aldrich, St. Louis, MO) at 1 ng/ml http://www.jimmunol.org/ (unless stated otherwise) or mouse IFN-b (PBL Biomedical Laboratories, In vivo LPS challenge New Brunswick, NJ) at 10 U/ml. rIL-10 (BD Biosciences, San Diego, CA) was used at 0.3 ng/ml. IFN-b depletion assays were performed by including Adult male mice (10-12 wk old) were injected i.p. with LPS (200 mg) and anti–IFN-b Ab (75-D3; Yamasa Soya, Tokyo, Japan) or control IgG at sacrificed 4 h later. Serum was collected, and IFN-b was measured by a concentration of 6.7 mg/ml. PD184352 (provided by Professor Sir Philip ELISA. Cohen, University of Dundee, U.K.) was used at a concentration of 2 mM. Retrovirally mediated gene expression Preparation of RNA For expression of TPL-2 and c-Fos, BMDM were infected with ecotropic retroviruses expressing the gene of interest and GFP, or GFP alone, as RNA was isolated in TRIzol (Invitrogen, Carlsbad, CA) or using RNeasy described (23). Following infection, GFP-positive BMDM were isolated by mini kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. by guest on October 1, 2021 FACS. Cells were cultured at 2 3 105 cells/well in 200 ml medium in a 96- Gene expression profiling well plate and stimulated with 10 ng/ml LPS for 24 h. The culture medium was used for IFN-b–specific ELISA, and cell pellets were lysed in 50 ml Probes were prepared from isolated RNA, and hybridized to Affymetrix lysis buffer (24) for measurement of total protein by Bradford assay (Bio- 430A 2.0 arrays. Two independent samples were analyzed for each condition. Rad). For expression of p105DN, BMDM were infected with retroviruses Expression data were read from CEL files, and background correction, expressing p105DN (murine p105 aa 434-971) and the gene for puromycin

Table I. Top 20 genes expressed at higher levels in Nfkb12/2Il102/2 BMDM than in Il102/2 BMDM following LPS stimulation

Symbol Name Fold-Change p Value Gbp1 Guanylate nucleotide-binding protein 1 182 0.00 Iigp1 IFN-inducible GTPase 1 19.0 0.01 Rsad2 Radical S-adenosyl methionine domain containing 2 14.2 0.00 Mx1 Myxovirus (influenza virus) resistance 1 14.0 0.01 Oasl1 29-59 Oligoadenylate synthetase-like 1 13.7 0.00 Tyki Thymidylate kinase family LPS-inducible member 12.6 0.01 Tgtp T cell-specific GTPase 12.2 0.01 Slfn1 Schlafen 1 12.2 0.02 Ifit2 IFN-induced protein with tetratricopeptide repeats 2 10.6 0.01 Mpa2l Macrophage activation 2-like 10.3 0.00 Il12b IL-12b 10.2 0.00 Usp18 Ubiquitin-specific peptidase 18 9.96 0.01 Gfi1 Growth factor independent 1 8.50 0.00 Il15 IL-15 7.24 0.00 Nt5c3 59-Nucleotidase, cytosolic III 7.01 0.00 Mx2 Myxovirus (influenza virus) resistance 2 6.89 0.00 Iigp2 IFN-inducible GTPase 2 6.29 0.00 Cxcl10 Chemokine (C-X-C motif) ligand 10 6.06 0.00 Chi3l1 Chitinase 3-like 1 5.82 0.00 Cxcl9 Chemokine (C-X-C motif) ligand 9 5.75 0.01 Fold-change refers to expression in Nfkb12/2Il102/2 BMDM divided by expression in Il102/2 BMDM following stim- ulation with LPS in the presence of exogenous IL-10. p refers to the p value between genotypes calculated using the Benjamini-Hochberg correction for multiple comparison testing. All listed genes were induced at least 4-fold by LPS in either Il102/2 or Nfkb12/2Il102/2 BMDM. Duplicates, expressed sequence tags, and hypothetical genes have been removed. The Journal of Immunology 3 resistance, or the gene for puromycin resistance alone. Puromcyin-resistant cells were isolated by selection for 72 h in media containing puromycin. After selection, cells were replated at 5 3 105 cells/well in 1 ml medium in a 24-well plate for total protein extraction, and at 5 3 104 cells/well in 200 ml media for analysis of cytokine secretion. Cells were stimulated the following day with LPS. Culture supernatants were assayed for IFN-b by ELISA. p50 DNA-binding ELISA p50 DNA-binding activity in nuclear extracts derived from LPS-stimulated WT and Map3k82/2 BMDM was measured with the p50 DNA-binding ELISA kit from Active Motif (Carlsbad, CA), as per the manufacturer’s instructions. Data are normalized to total protein in the nuclear extracts determined by Bradford assay (Bio-Rad). Statistical analysis All data analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA). Data generated by RT-PCR or ELISA were compared using t tests for parametric data; *p , 0.05, **p , 0.01, and ***p , 0.001. Error bars represent SEM.

Results Downloaded from Profiling LPS-induced gene expression in p50/p105-deficient macrophages To characterize the role of p50/p105 in regulating inflammatory gene expression, we profiled LPS-induced gene expression in BMDM lacking p50/p105 and IL-10 (Nfkb12/2Il102/2). Experi- ments were performed with BMDM additionally lacking IL-10 http://www.jimmunol.org/ because LPS-induced expression of endogenous IL-10 has a strong inhibitory effect on the expression of many inflammatory genes (25), and there are marked differences in levels of IL- 10 secretion following TLR stimulation of WT and p50/p105- deficient macrophages (24, 26, 27). Low levels of exogenous IL-10 were added to cultures to amplify differences in expression between Il102/2 and Nfkb12/2Il102/2 BMDM (20).

Compared with expression in unstimulated cells, 216 of 22,626 by guest on October 1, 2021 probe sets evaluated were induced at least 4-fold by LPS in either Il102/2 or Nfkb12/2Il102/2 BMDM (Supplemental Table I). Expression levels were significantly higher for 121 of these 216 LPS-induced probe sets in Nfkb12/2Il102/2 compared with Il102/2 BMDM, whereas 16 were expressed at significantly lower levels. The 20 genes with the largest fold-change between LPS stimulated Nfkb12/2Il102/2 and Il102/2 BMDM are shown in Table I. A significant fraction of these were type I IFN-responsive genes, including Mx1 and Cxcl9 (28, 29). This suggested that p50/p105 might preferentially inhibit expression of IFN-responsive genes following LPS stimulation. FIGURE 1. Effect of p50/p105 deficiency on LPS-induced gene ex- pression. A, Fold induction as determined by RT-PCR of IFN-responsive p50/p105 inhibits TLR-induced IFN-responsive gene genes 4 h after stimulation with LPS. B, Fold induction of IFN-non- expression responsive genes 4 h after stimulation with LPS. C, Fold induction of To determine whether p50/p105 deficiency selectively increased indicated genes 4 h after stimulation with IFN-b. Please note log scale for IFN-responsive gene expression in the absence of exogenous IL- y-axis. D, Fold induction of Ifnb1 mRNA (left) or amount of IFN-b protein accumulation in the medium as determined by ELISA (right), 4 and 24 h 10, we next analyzed a select set of LPS-inducible genes by RT- after stimulation with LPS, respectively. Each column represents the mean PCR. These genes included three that have previously been report- of three independent macrophage pools for each genotype. One of two ed as IFN responsive (Mx1, Cxcl9, and Nos2) (28–30), and three experiments with similar results. that are not IFN responsive (Cxcl1, Cxcl2, and Il1b). These experiments were performed in BMDM that also lack the gene for 2 2 2 2 2 2 2 2 RAG2, as we found that lymphocyte-sufficient mice lacking both to comparable levels in Nfkb1 / Il10 / Rag2 / and Il10 / 2 2 p50 and IL-10 spontaneously develop inflammatory disease as Rag2 / BMDM following stimulation with exogenous IFN-b, they age (B.H. Horwitz, unpublished data). Consistent with the whereas Cxcl1, Cxcl2, and Il1b were not induced in cells of either gene-profiling data, mRNAs encoding IFN-responsive genes were genotype (Fig. 1C). These results confirmed that p50/p105 sup- expressed at significantly higher levels in Nfkb12/2Il102/2Rag22/2 pressed the ability of LPS to induce expression of IFN-responsive BMDM than in Il102/2Rag22/2 BMDM after LPS stimula- genes, and also demonstrated that p50/p105 did not regulate the tion (Fig. 1A), whereas the mRNAs encoding genes that are not sensitivity of macrophages to IFN-b stimulation. Furthermore, IFN responsive were expressed at similar or lower levels (Fig. these data indicated that p50/p105 had inhibitory functions that 1B). Expression of Mx1, Cxcl9, and Nos2 mRNAs was induced were independent of the regulation of, or response to, IL-10. 4 NF-kB1 INHIBITS TLR-INDUCED IFN SIGNALING

We next determined whether the increase in IFN-responsive gene elevated expression of IFN-dependent genes in macrophages lack- expression observed following LPS stimulation of BMDM lacking ing p50/p105 depended on autocrine IFN-b stimulation and aug- p50/p105 could result from increased production of IFN-b itself. mented activation of STAT1. These data are consistent with Consistent with this hypothesis, we found that LPS induced sig- a recent report showing increased LPS-induced IFN-b and IFN- nificantly higher expression of Ifnb1 mRNA, as well as higher dependent signaling in LPS-stimulated peritoneal exudative cells levels of secreted IFN-b protein, in Nfkb12/2Il102/2Rag22/2 isolated from Nfkb12/2 mice (9). 2/2 2/2 BMDM than in Il10 Rag2 BMDM (Fig. 1D). 2 2 LPS induces increased serum levels of IFN-b in Nfkb1 / mice Increased IFN signaling observed in the absence of p50/p105 To determine whether p50/p105 suppresses the induction of IFN-b b depends on expression of IFN- following inflammatory challenge in vivo, WT and Nfkb12/2 mice The biological effects of IFN-b are mediated largely through ac- on the C57BL/6 background received 200 mg LPS i.p. Serum was tivation of STAT1. LPS-induced phosphorylation of STAT1 was harvested 4 h later, and levels of IFN-b were determined by significantly augmented in the absence of p50/p105 (Fig. 2A), and ELISA. LPS induced detectable levels of IFN-b in the serum of this was markedly inhibited by treatment of Nfkb12/2Il102/2 Nfkb12/2 mice but not in WT mice (Fig. 3). This demonstrates Rag22/2 BMDM with an IFN-b–blocking Ab (31) (Fig. 2B). that p50/p105 suppresses the systemic induction of IFN-b fol- Ab treatment also impaired LPS-induced expression of IFN- lowing LPS challenge. responsive genes Cxcl9 and Nos2, while having little effect on Inhibition of TLR-induced IFN signaling by p50/p105 depends expression of IFN-b itself (Fig. 2C). These results suggest that on TPL-2 2 2 LPS-induced ERK activation is blocked in Nfkb1 / BMDM due Downloaded from to TPL-2 deficiency (11, 17, 32), and we have recently shown that the TPL-2/ERK signaling pathway negatively regulates LPS in- duction of IFN-b in macrophages (24). This raised the possibility that defective TPL-2/ERK signaling was responsible for increased IFN-b signaling observed in p50/p105-deficient BMDM. Indeed,

in addition to the expected increase in IFN-b expression (Sup- http://www.jimmunol.org/ plemental Fig. 1A), we detected increased STAT1 phosphorylation (Fig. 4A) and increased IFN-dependent gene expression (Fig. 4B) in TPL-2–deficient (Map3k82/2) BMDM compared with WT BMDM following LPS stimulation. Therefore, the phenotype of TPL-2–deficient Map3k82/2 macrophages was very similar to that of p50/p105-deficient macrophages. To investigate whether the effect of TPL-2 deficiency on type I IFN signaling was due to impaired activation of ERK, WT BMDM were pretreated with the MEK inhibitor PD184352 prior to sti- by guest on October 1, 2021 mulation with LPS. Blockade of ERK activation with PD184352 (Fig. 5A) increased STAT1 phosphorylation (Fig. 5B) and IFN- responsive gene expression (Fig. 5C), as well as increasing ex- pression of IFN-b, as previously described (24) (Supplemental Fig. 1B). Therefore, the effects of TPL-2 deficiency on IFN sig- naling were phenocopied by pharmacological blockade of ERK activation, consistent with the critical role of TPL-2 in regulating ERK activation in TLR-stimulated macrophages (Fig. 4A).

FIGURE 2. p50/p105 deficiency increases phosphorylation of STAT1 following TLR stimulation. A, Immunoblot of p-STAT1 (Y701) and total STAT1 in cell extracts from Il102/2Rag2/2 and Nfkb12/2Il102/2Rag2/2 BMDM following stimulation with LPS at indicated time points. ERK is included as a loading control. Numbers under the p-STAT1 band indicate the intensity of the p-STAT1 band relative to the intensity in the unstimulated Il102/2Rag2/2 sample, normalized to total STAT1. B, Immunoblot of p- STAT1 (Y701) and total STAT1 in nuclear extracts from Nfkb12/2Il102/2 Rag2/2 BMDM 4 h after stimulation with LPS in the presence or absence of IFN-b–specific depleting Ab. TFIID is included as a nuclear loading control. C, Fold induction of indicated IFN-responsive genes in Nfkb12/2Il102/2 FIGURE 3. Increased levels of IFN-b in the serum of Nfkb12/2 mice Rag2/2 BMDM 4 h after stimulation with LPS in the presence or absence after LPS challenge. WT and Nfkb12/2 mice received 200 mg LPS, and of an IFN-b–blocking Ab. Columns represent mean values of three in- serum was harvested 4 h later. IFN-b levels were measured in the serum dependent macrophage pools for each genotype. One of two experiments by ELISA. Each column represents five mice per genotype. One of two with similar results. experiments with similar results. The Journal of Immunology 5

FIGURE 4. Increased IFN signaling in TPL-2– deficient BMDM. A, Immunoblotting of p-STAT1 (Y701), total STAT1, p-ERK 1/2, or total ERK in WT, Nfkb12/2,orMap3k82/2 BMDM at in- dicated times following stimulation with LPS. B, Fold induction of Cxcl9 and Nos2 in WT or Map3k82/2 BMDM 4 h after stimulation with LPS. Each column represents two independent mice per genotype. One of two experiments with similar results. Downloaded from http://www.jimmunol.org/ To test the hypothesis that decreased levels of TPL-2 in with control virus (Fig. 6A). These results indicate that impaired p50/p105-deficient BMDM were responsible for enhanced ex- activation of the TPL-2/ERK signaling pathway in p50/p105- pression of Ifnb1 following LPS stimulation, p50/p105-deficient deficient BMDM is responsible for increased IFN signaling ob- BMDM were infected with a retrovirus encoding both TPL-2 and served in these cells. GFP, to increase TPL-2 protein levels (23), or control vector It has been previously shown that the C-terminal ankyrin repeat encoding GFP alone. After sorting for GFP-positive cells, BMDM region of p105 is necessary to stabilize TPL-2 and facilitate ERK were stimulated with LPS, and the production of IFN-b was activation in LPS-stimulated macrophages (11, 17, 33). Therefore, evaluated by ELISA. Infection with the virus expressing TPL-2 our results imply that the C-terminal region of p105 has essential by guest on October 1, 2021 markedly inhibited induction of IFN-b compared with infection functions necessary to inhibit Ifnb1 expression that are inde- pendent of any putative inhibitory function of p50 homodimers. Consistent with this hypothesis, TPL-2–deficient Map3k82/2 BMDM exhibit normal levels of active p50 after LPS stimulation, as detected by NF-kB ELISA (Fig. 6B). Furthermore, the obser- vation that Map3k82/2 and Nfkb12/2 macrophages produce sim- ilar levels of IFN-b after LPS stimulation (Fig. 6C) suggests that the role of p50 homodimers may be relatively minor. To directly investigate whether the C-terminal ankyrin repeat region of p105 is responsible for inhibition of IFN-b expression, we infected BMDM derived from Nfkb12/2Il102/2Rag2/2 mice with either a control retrovirus (vector) or one expressing the C- terminal ankyrin repeat domain of p105 (p105DN) tagged with hemagglutinin. Infected BMDM were selected with puromycin, and following replating at equal density, were stimulated with LPS. Immunoblotting of BMDM lysates confirmed successful expression of p105DN (Fig. 7A), which rescued activation or ERK following LPS stimulation, as expected (Fig. 7A). Furthermore, in response to LPS, p105DN markedly inhibited secretion of IFN-b compared with cells infected with control vector (Fig. 7B). These results show that the C-terminal ankyrin repeat region of p105 inhibits induction of IFN-b secretion following LPS challenge. c-Fos is a member of the AP-1 transcription factor family that is induced in response to TLR ligation (34). Induction of c-Fos FIGURE 5. ERK inhibition augments LPS-induced IFN signaling. A, depends on ERK activation, and it has been demonstrated that Immunoblotting of p-ERK and total ERK in total cell extracts of WT 6 B c-Fos can inhibit expression of IFN-b (24). c-Fos DNA-binding BMDM stimulated with LPS for 30 min PD184352 (PD). , Immu- 2/2 noblotting of p-STAT1 (Y701), total STAT1, and total ERK in WT BMDM activity is decreased following TLR stimulation of Map3k8 stimulated for 2 h 6 PD. C, Fold induction of Cxcl9 and Nos2 mRNA in BMDM compared with WT cells (24), and defective induction of WT BMDM stimulated for 4 h 6 PD. Representative of three similar c-Fos protein expression has been observed following stimulation 2 2 experiments. of p50/p105-deficient Nfkb1 / BMDMs with Helicobacter 6 NF-kB1 INHIBITS TLR-INDUCED IFN SIGNALING Downloaded from

FIGURE 7. Expression of p105DN rescues ERK activation, and inhibits IFN-b secretion in Nfkb12/2 BMDM. A, Il102/2Rag2/2 and Nfkb12/2 Il102/2Rag2/2 BMDM infected with the vector alone (vector) or the vector encoding p105DN (p105DN) were lysed following stimulation with LPS, and immunoblotted for the indicated Ags. B, IFN-b secretion in http://www.jimmunol.org/ culture supernatants was measured by ELISA 12 h after LPS stimulation in Il102/2Rag2/2 and Nfkb12/2Il102/2Rag2/2 BMDM infected with the vector alone, or with p105DN. Each column represents the mean of three independent macrophage pools.

phages lacking TPL-2 also exhibit increased STAT1 activation and FIGURE 6. Expression of TPL-2 inhibits LPS-induced IFN-b pro- IFN-dependent gene expression following LPS stimulation, and duction by Nfkb12/2 macrophages. A, Accumulation of IFN-b in medium + 2/2 2/2 that this is phenocopied by inhibition of ERK. These data are 24 h after LPS stimulation of sorted GFP Map3k8 and Nfkb1 by guest on October 1, 2021 BMDM, retrovirally infected with either control GFP vector (vector) or consistent with our earlier demonstration that the TPL-2/ERK vector encoding GFP and TPL-2. Levels of IFN-b were measured by signaling pathway negatively regulates TLR-induced IFN-b pro- ELISA and normalized to total cellular protein. B, p50 DNA-binding ac- duction by macrophages (24). Retroviral expression of TPL-2 in 2/2 tivity in WT or Map3k82/2 BMDM measured by NF-kB DNA-binding Nfkb1 BMDM, which are deficient in endogenous TPL-2, ELISA. C, Accumulation of IFN-b in the medium 24 h after stimulation of inhibits LPS-induced induction of IFN-b, indicating that im- 2 2 2 2 WT, Map3k8 / , and Nfkb1 / BMDM with LPS. Each column repre- paired TPL-2–dependent ERK activation is responsible for sents mean of two mice. One of two experiments with similar results. hepaticus (32). Consistent with these previous reports, we ob- served a defect in the expression of c-Fos protein following LPS stimulation of Nfkb12/2 and Map3k82/2 BMDM (Fig. 8A). Fur- thermore, retroviral-mediated expression of c-Fos significantly inhi- bited IFN-b LPS-induced IFN-b secretion in Nfkb12/2 BMDM (Fig. 8B). These results indicate that reduced induction of c-Fos following LPS stimulation may be responsible for augmented production of IFN-b by p50/p105-deficient macrophages.

Discussion The results reported in this study demonstrate that p50/p105 suppresses the ability of LPS to induce IFN-b expression both in macrophages in vitro and in vivo following i.p. injection. In- creased IFN-responsive gene expression observed in the absence of p50/p105 is, at least in part, dependent on expression of IFN-b, FIGURE 8. Expression of c-Fos downregulates IFN-b expression in as IFN-b depletion interferes with expression of IFN-responsive 2 2 Nfkb1 / BMDM. A, Immunoblot of c-Fos in total cell extracts from WT, genes in p50/p105-deficient BMDM. These results are consistent Nfkb12/2, and Map3k82/2 BMDM stimulated with LPS for indicated with a recently published study demonstrating increased expres- times. B, IFN-b secretion following LPS stimulation of sorted GFP+ sion of IFN-b, Cxcl9, and Nos2, as well as increased STAT1 ac- Nfkb12/2 BMDM infected with the vector alone or infected with the vector tivation in LPS-treated peritoneal exudative cells harvested from encoding c-Fos. Each column represents three independent mice per group. mice lacking p50/p105 (9). We have further shown that macro- One of two experiments with similar results. The Journal of Immunology 7 increased IFN expression and IFN signaling observed in BMDM periments further suggest that LPS-induced expression of c-Fos, lacking p50/p105. Expression of the C-terminal ankyrin repeat which depends on the p105/TPL-2/ERK pathway, inhibits IFN-b domain of p105 in Nfkb12/2 macrophages, which reconstitutes expression, providing a mechanism for the increase in IFN-b LPS-induced ERK activation, also suppresses IFN-b secretion, expression observed in Nfkb12/2 macrophages. consistent with the role of the C-terminal domain in stabilizing the In conclusion, this study shows that a major function for NF-kB1 steady-state expression of TPL-2. Finally, expression of c-Fos p50/p105 in regulating gene expression in TLR-stimulated mac- in Nfkb12/2 BMDM inhibits IFN-b secretion, suggesting that rophages is to facilitate activation of the TPL-2/ERK signaling the reduction in ERK-dependent c-Fos expression observed in pathway by maintaining steady-state TPL-2 protein levels. These Nfkb12/2 BMDM may be an important factor leading to elevated data raise the possibility that in vivo phenotypes of Nfkb12/2 mice production of IFN-b. that have been attributed to altered NF-kB function might actually IL-10 expression is reduced in macrophages lacking p50/p105 be due, at least in part, to impaired activation of ERK MAPK in compared with WT cells after TLR stimulation. Because IL-10 innate immune responses. This is supported by the observation is a potent inhibitor of many TLR-inducible genes, including that mice lacking only p105 are susceptible to colitis (38), similar IFN-b and CXCL9 (35), it is possible that increased expression to the colitis-susceptible phenotype previously observed in mice of IFN-responsive genes observed following LPS stimulation of lacking both p50 and p105 (18, 20, 27). Further studies will be Nfkb12/2 BMDM could in part result from decreased production necessary to define the relative importance of impaired activation of IL-10. However, as our initial experiments were performed of the TPL-2/ERK pathway versus altered NF-kB activation for using macrophages that also lack the ability to secrete IL-10, the phenotype of Nfkb12/2 mice in models of infectious disease differences in gene expression observed in this study could not and autoimmunity. be solely due to altered endogenous production of IL-10. p50/ Downloaded from p105 therefore has inhibitory functions that are independent of Disclosures IL-10. Consistent with this conclusion, we previously demon- The authors have no financial conflicts of interest. strated that TPL-2/ERK signaling negatively regulates IFN-b production in the absence of IL-10 (24). It has been reported that macrophages lacking IKK-b exhibited References augmented activation of STAT1 following stimulation with heat- 1. Grilli, M., J. J. Chiu, and M. J. Lenardo. 1993. NF-kappa B and Rel: participants http://www.jimmunol.org/ in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143: 1–62. killed group B Streptococcus (36). Because IKK-b regulates ac- 2. Ghosh, S., M. J. May, and E. B. Kopp. 1998. NF-kappa B and Rel proteins: tivation of the TPL-2/ERK signaling pathway via phosphorylation evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16: 225–260. of p105 (11, 13), it might be expected that increased STAT1 ac- 3. Rothwarf, D. M., and M. Karin. 1999. The NF-kappa B activation pathway: tivation in IKK-b–deficient macrophages resulted from aug- a paradigm in information transfer from membrane to nucleus. Sci. STKE 1999: mented IFN-b production, as in Nfkb12/2 macrophages. Surpri- RE1. 4. Li, Q., and I. M. Verma. 2002. NF-kappaB regulation in the immune system. Nat. singly, however, IFN-b production was reported to be reduced Rev. Immunol. 2: 725–734. in the absence of IKK-b (36). These data suggest that IKK-b 5. Kang, S. M., A. C. Tran, M. Grilli, and M. J. Lenardo. 1992. NF-kappa B subunit most likely has roles in both the induction, and p105-dependent regulation in nontransformed CD4+ T lymphocytes. Science 256: 1452–1456. 6. Zhong, H., M. J. May, E. Jimi, and S. Ghosh. 2002. The phosphorylation status by guest on October 1, 2021 inhibition, of IFN-b expression following LPS stimulation. The of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1. observation that LPS-induced IFN-b expression is lower in mac- Mol. Cell 9: 625–636. rophages lacking IKK-b suggests that any decrease in activation 7. Bohuslav, J., V. V. Kravchenko, G. C. Parry, J. H. Erlich, S. Gerondakis, N. Mackman, and R. J. Ulevitch. 1998. Regulation of an essential innate immune of the p105-dependent inhibitory pathway does not overcome the response by the p50 subunit of NF-kappaB. J. Clin. Invest. 102: 1645–1652. reduction in the primary IKK/NF-kB–dependent induction of 8. Kastenbauer, S., and H. W. Ziegler-Heitbrock. 1999. NF-kappaB1 (p50) is up- IFN-b transcription. The mechanism for the negative regulation of regulated in lipopolysaccharide tolerance and can block tumor necrosis factor gene expression. Infect. Immun. 67: 1553–1559. STAT1 activation by IKK-b, which is apparently independent 9. Porta, C., M. Rimoldi, G. Raes, L. Brys, P. Ghezzi, D. Di Liberto, F. Dieli, of effects on IFN-b production, to our knowledge, remains un- S. Ghisletti, G. Natoli, P. De Baetselier, et al. 2009. Tolerance and M2 (alter- native) macrophage polarization are related processes orchestrated by p50 nu- known. clear factor kappaB. Proc. Natl. Acad. Sci. USA 106: 14978–14983. Macrophages can exhibit divergent functional programs de- 10. Saccani, A., T. Schioppa, C. Porta, S. K. Biswas, M. Nebuloni, L. Vago, pending on the nature of the activating stimuli (37). Classic or B. Bottazzi, M. P. Colombo, A. Mantovani, and A. Sica. 2006. p50 nuclear factor-kappaB overexpression in tumor-associated macrophages inhibits M1 M1 activation is induced by microbial products and/or IFN- inflammatory responses and antitumor resistance. Cancer Res. 66: 11432–11440. g, which induce a proinflammatory response (increased IL-12, 11. Beinke, S., M. J. Robinson, M. Hugunin, and S. C. Ley. 2004. Lipopolysac- NOS2, MHC class II, and IFN-b expression) required to kill in- charide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IkappaB kinase- tracellular pathogens. In contrast, M2 alternative activation by induced proteolysis of NF-kappaB1 p105. Mol. Cell. Biol. 24: 9658–9667. IL-4 and IL-13 following parasite infection is characterized by 12. Lang, V., J. Janzen, G. Z. Fischer, Y. Soneji, S. Beinke, A. Salmeron, H. Allen, decreased expression of NOS2 and MHC class II, and increased R. T. Hay, Y. Ben-Neriah, and S. C. Ley. 2003. betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932. Mol. expression of anti-inflammatory cytokines such as IL-10. Based Cell. Biol. 23: 402–413. 2 2 on analyses of Nfkb1 / macrophages, Porta et al. (9) have pro- 13. Waterfield, M., W. Jin, W. Reiley, M. Zhang, and S. C. Sun. 2004. IkappaB posed that p50 inhibits M1 polarization of LPS-stimulated mac- kinase is an essential component of the Tpl2 signaling pathway. Mol. Cell. Biol. 24: 6040–6048. rophages. As overexpression of p50 in RAW 267.4 macrophages 14. Sriskantharajah, S., M. P. Belich, S. Papoutsopoulou, J. Janzen, V. Tybulewicz, suppressed LPS-induced IFN-b promoter activity, it was sug- B. Seddon, and S. C. Ley. 2009. Proteolysis of NF-kappaB1 p105 is essential for b T cell antigen receptor-induced proliferation. Nat. Immunol. 10: 38–47. gested that p50 homodimers might directly inhibit IFN- tran- 15. Belich, M. P., A. Salmero´n, L. H. Johnston, and S. C. Ley. 1999. TPL-2 kinase scription (9). Our study indicates that the C-terminal ankyrin regulates the proteolysis of the NF-kappaB-inhibitory protein NF-kappaB1 p105. repeat domain of p105, which is known to interact with and sta- Nature 397: 363–368. 16. Dumitru, C. D., J. D. Ceci, C. Tsatsanis, D. Kontoyiannis, K. Stamatakis, bilize TPL-2 (17), inhibits IFN-b expression by facilitating LPS- J. H. Lin, C. Patriotis, N. A. Jenkins, N. G. Copeland, G. Kollias, and induced ERK activation. These results are consistent with the P. N. Tsichlis. 2000. TNF-alpha induction by LPS is regulated posttranscrip- hypothesis that defective TPL-2/ERK signaling is the predominant tionally via a Tpl2/ERK-dependent pathway. Cell 103: 1071–1083. 17. Waterfield, M. R., M. Zhang, L. P. Norman, and S. C. Sun. 2003. NF-kappaB1/ factor leading to elevated expression of IFN-b in p50/p105- p105 regulates lipopolysaccharide-stimulated MAP kinase signaling by gov- deficient macrophages, rather than the absence of p50. Our ex- erning the stability and function of the Tpl2 kinase. Mol. Cell 11: 685–694. 8 NF-kB1 INHIBITS TLR-INDUCED IFN SIGNALING

18. Erdman, S., J. G. Fox, C. A. Dangler, D. Feldman, and B. H. Horwitz. 2001. 29. Amichay, D., R. T. Gazzinelli, G. Karupiah, T. R. Moench, A. Sher, and Typhlocolitis in NF-kappa B-deficient mice. J. Immunol. 166: 1443–1447. J. M. Farber. 1996. Genes for chemokines MuMig and Crg-2 are induced in 19. Sha, W. C., H. C. Liou, E. I. Tuomanen, and D. Baltimore. 1995. Targeted protozoan and viral infections in response to IFN-gamma with patterns of tissue disruption of the p50 subunit of NF-kappa B leads to multifocal defects in im- expression that suggest nonredundant roles in vivo. J. Immunol. 157: 4511–4520. mune responses. Cell 80: 321–330. 30. Vadiveloo, P. K., G. Vairo, P. Hertzog, I. Kola, and J. A. Hamilton. 2000. Role of 20. Tomczak, M. F., S. E. Erdman, A. Davidson, Y. Y. Wang, P. R. Nambiar, type I interferons during macrophage activation by lipopolysaccharide. Cytokine A. B. Rogers, B. Rickman, D. Luchetti, J. G. Fox, and B. H. Horwitz. 2006. 12: 1639–1646. Inhibition of Helicobacter hepaticus-induced colitis by IL-10 requires the p50/ 31. Toshchakov, V., B. W. Jones, P. Y. Perera, K. Thomas, M. J. Cody, S. Zhang, p105 subunit of NF-kappa B. J. Immunol. 177: 7332–7339. B. R. Williams, J. Major, T. A. Hamilton, M. J. Fenton, and S. N. Vogel. 2002. 21. Wang, Y., B. H. Rickman, T. Poutahidis, K. Schlieper, E. A. Jackson, TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent S. E. Erdman, J. G. Fox, and B. H. Horwitz. 2008. c-Rel is essential for the gene expression in macrophages. Nat. Immunol. 3: 392–398. development of innate and T cell-induced colitis. J. Immunol. 180: 8118–8125. 32. Tomczak, M. F., M. Gadjeva, Y. Y. Wang, K. Brown, I. Maroulakou, 22. Wettenhall, J. M., K. M. Simpson, K. Satterley, and G. K. Smyth. 2006. P. N. Tsichlis, S. E. Erdman, J. G. Fox, and B. H. Horwitz. 2006. Defective affylmGUI: a graphical user interface for linear modeling of single channel activation of ERK in macrophages lacking the p50/p105 subunit of NF-kappaB microarray data. Bioinformatics 22: 897–899. is responsible for elevated expression of IL-12 p40 observed after challenge with 23. Robinson, M. J., S. Beinke, A. Kouroumalis, P. N. Tsichlis, and S. C. Ley. 2007. Helicobacter hepaticus. J. Immunol. 176: 1244–1251. Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide ac- 33. Beinke, S., J. Deka, V. Lang, M. P. Belich, P. A. Walker, S. Howell, tivation of extracellular signal-regulated kinase in macrophages. Mol. Cell. Biol. S. J. Smerdon, S. J. Gamblin, and S. C. Ley. 2003. NF-kappaB1 p105 negatively 27: 7355–7364. regulates TPL-2 MEK kinase activity. Mol. Cell. Biol. 23: 4739–4752. 24. Kaiser, F., D. Cook, S. Papoutsopoulou, R. Rajsbaum, X. Wu, H. T. Yang, 34. Introna, M., T. A. Hamilton, R. E. Kaufman, D. O. Adams, and R. C. Bast, Jr. S. Grant, P. Ricciardi-Castagnoli, P. N. Tsichlis, S. C. Ley, and A. O’Garra. 2009. TPL-2 negatively regulates interferon-beta production in macrophages and 1986. Treatment of murine peritoneal macrophages with bacterial lipopolysac- myeloid dendritic cells. J. Exp. Med. 206: 1863–1871. charide alters expression of c-fos and c-myc oncogenes. J. Immunol. 137: 2711– 25. Lang, R., D. Patel, J. J. Morris, R. L. Rutschman, and P. J. Murray. 2002. 2715. Shaping gene expression in activated and resting primary macrophages by IL-10. 35. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. J. Immunol. 169: 2253–2263. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19: 683– 26. Cao, S., X. Zhang, J. P. Edwards, and D. M. Mosser. 2006. NF-kappaB1 (p50) 765. Downloaded from homodimers differentially regulate pro- and anti-inflammatory cytokines in 36. Fong, C. H., M. Bebien, A. Didierlaurent, R. Nebauer, T. Hussell, D. Broide, macrophages. J. Biol. Chem. 281: 26041–26050. M. Karin, and T. Lawrence. 2008. An antiinflammatory role for IKKbeta through 27. Tomczak, M. F., S. E. Erdman, T. Poutahidis, A. B. Rogers, H. Holcombe, the inhibition of “classical” macrophage activation. J. Exp. Med. 205: 1269– B. Plank, J. G. Fox, and B. H. Horwitz. 2003. NF-kappa B is required within the 1276. innate immune system to inhibit microflora-induced colitis and expression of IL- 37. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of mac- 12 p40. J. Immunol. 171: 1484–1492. rophage activation. Nat. Rev. Immunol. 8: 958–969. 28. Hug, H., M. Costas, P. Staeheli, M. Aebi, and C. Weissmann. 1988. Organization 38. Chang, M., A. J. Lee, L. Fitzpatrick, M. Zhang, and S. C. Sun. 2009. NF-kappa

of the murine Mx gene and characterization of its interferon- and virus-inducible B1 p105 regulates T cell homeostasis and prevents chronic inflammation. J. http://www.jimmunol.org/ promoter. Mol. Cell. Biol. 8: 3065–3079. Immunol. 182: 3131–3138. by guest on October 1, 2021 Table S1. Fold change between Nfkb1-/-Il10-/- and Il10-/- BMDM for all 216 probe sets induced at least 4-fold by LPS.

Affy ID Symbol Name FC p 1420549_at Gbp1 guanylate nucleotide binding protein 1 182.13 0.00 1419043_a_at Iigp1 interferon inducible GTPase 1 19.01 0.01 1421008_at Rsad2 radical S-adenosyl methionine domain containing 2 14.27 0.00 1451905_a_at Mx1 myxovirus (influenza virus) resistance 1 14.03 0.01 1424339_at Oasl1 2'-5' oligoadenylate synthetase-like 1 13.77 0.00 1419042_at Iigp1 interferon inducible GTPase 1 13.01 0.01 1422095_a_at Tyki thymidylate kinase family LPS-inducible member 12.68 0.01 1449009_at Tgtp T-cell specific GTPase 12.30 0.01 1418612_at Slfn1 schlafen 1 12.23 0.02 1418293_at Ifit2 interferon-induced protein with tetratricopeptide 10.68 0.01 repeats 2 1435331_at AI447904 expressed sequence AI447904 10.44 0.02 1421009_at Rsad2 radical S-adenosyl methionine domain containing 2 10.40 0.00 1438676_at Mpa2l macrophage activation 2 like 10.36 0.00 1449497_at Il12b interleukin 12b 10.21 0.00 1418191_at Usp18 ubiquitin specific peptidase 18 9.97 0.01 1450484_a_at Tyki thymidylate kinase family LPS-inducible member 9.67 0.00 1419530_at Il12b interleukin 12b 9.13 0.00 1417679_at Gfi1 growth factor independent 1 8.50 0.00 1418219_at Il15 interleukin 15 7.25 0.00 1451050_at Nt5c3 5'-nucleotidase, cytosolic III 7.01 0.00 1419676_at Mx2 myxovirus (influenza virus) resistance 2 6.90 0.00 1431591_s_at LOC677168 hypothetical protein LOC677168 6.87 0.01 1428027_at NA NA 6.62 0.00 1438716_at AI451617 expressed sequence AI451617 6.33 0.00 1417793_at Iigp2 interferon inducible GTPase 2 6.30 0.00 1418930_at Cxcl10 chemokine (C-X-C motif) ligand 10 6.06 0.00 1451537_at Chi3l1 chitinase 3-like 1 5.82 0.00 1418652_at Cxcl9 chemokine (C-X-C motif) ligand 9 5.76 0.01 1430005_a_at Batf2 basic leucine zipper transcription factor, ATF-like 2 5.49 0.00 1425806_a_at Surb7 SRB7 (suppressor of RNA polymerase B) homolog (S. 5.38 0.00 cerevisiae) 1450783_at Ifit1 interferon-induced protein with tetratricopeptide 5.16 0.01 repeats 1 1450297_at Il6 interleukin 6 5.02 0.00 1419026_at Daxx Fas death domain-associated protein 4.93 0.00 1453939_x_at LOC677168 hypothetical protein LOC677168 4.84 0.00 1417292_at Ifi47 interferon gamma inducible protein 47 4.83 0.01 1449227_at Ch25h cholesterol 25-hydroxylase 4.83 0.00 1417141_at Igtp interferon gamma induced GTPase 4.72 0.00 1436058_at Rsad2 radical S-adenosyl methionine domain containing 2 4.60 0.01 1425156_at Gbp6 guanylate binding protein 6 4.55 0.00 1425394_at BC023105 cDNA sequence BC023105 4.50 0.00 1450403_at Stat2 signal transducer and activator of transcription 2 4.46 0.01 1419697_at Cxcl11 chemokine (C-X-C motif) ligand 11 4.38 0.07 1450173_at Ripk2 receptor (TNFRSF)-interacting serine-threonine kinase 4.22 0.00 2 1448940_at Trim21 tripartite motif protein 21 4.19 0.00 1419149_at Serpine1 serine (or cysteine) peptidase inhibitor, clade E, 4.11 0.00 member 1 1450788_at Saa1 serum amyloid A 1 4.10 0.00 1449450_at Ptges prostaglandin E synthase 4.08 0.00 1418317_at Lhx2 LIM homeobox protein 2 3.99 0.01 1421679_a_at Cdkn1a cyclin-dependent kinase inhibitor 1A (P21) 3.97 0.00 1450446_a_at Socs1 suppressor of cytokine signaling 1 3.97 0.01 1421911_at Stat2 signal transducer and activator of transcription 2 3.88 0.00 1418077_at Trim21 tripartite motif protein 21 3.85 0.00 1421473_at Il1a interleukin 1 alpha 3.82 0.00 1419604_at Zbp1 Z-DNA binding protein 1 3.73 0.02 1419858_at Ifi203 interferon activated gene 203 3.69 0.00 1450639_at Slc28a2 solute carrier family 28 (sodium-coupled nucleoside 3.66 0.01 transporter), member 2 1451340_at Arid5a AT rich interactive domain 5A (Mrf1 like) 3.58 0.00 1451567_a_at Ifi203 interferon activated gene 203 3.53 0.01 1436899_at 2700019D07Rik RIKEN cDNA 2700019D07 gene 3.43 0.00 1449449_at Ptges prostaglandin E synthase 3.43 0.00 1452178_at Plec1 plectin 1 3.34 0.00 1435454_a_at BC006779 cDNA sequence BC006779 3.34 0.01 1427736_a_at Ccrl2 chemokine (C-C motif) receptor-like 2 3.32 0.00 1434372_at AW112010 expressed sequence AW112010 3.07 0.05 1421236_at Ripk2 receptor (TNFRSF)-interacting serine-threonine kinase 2.99 0.00 2 1424698_s_at Gca grancalcin 2.97 0.00 1449028_at Rhou ras homolog gene family, member U 2.95 0.00 1418392_a_at Gbp3 guanylate nucleotide binding protein 3 2.94 0.00 1426276_at Ifih1 interferon induced with helicase C domain 1 2.89 0.00 1419282_at Ccl12 chemokine (C-C motif) ligand 12 2.85 0.09 1434380_at Gbp6 guanylate binding protein 6 2.83 0.00 1424638_at Cdkn1a cyclin-dependent kinase inhibitor 1A (P21) 2.82 0.00 1448436_a_at Irf1 interferon regulatory factor 1 2.81 0.00 1460415_a_at Cd40 CD40 antigen 2.78 0.00 1449473_s_at Cd40 CD40 antigen 2.77 0.00 1429527_a_at Plscr1 phospholipid scramblase 1 2.76 0.00 1420499_at Gch1 GTP cyclohydrolase 1 2.73 0.00 1453181_x_at Plscr1 phospholipid scramblase 1 2.70 0.00 1436172_at Samd9l sterile alpha motif domain containing 9-like 2.52 0.02 1450672_a_at Trex1 three prime repair exonuclease 1 2.48 0.02 1419721_at Gpr109a G protein-coupled receptor 109A 2.46 0.01 1417262_at Ptgs2 prostaglandin-endoperoxide synthase 2 2.33 0.00 1449027_at Rhou ras homolog gene family, member U 2.27 0.01 1419714_at Cd274 CD274 antigen 2.26 0.00 1417851_at Cxcl13 chemokine (C-X-C motif) ligand 13 2.26 0.34 1455393_at Cp ceruloplasmin 2.25 0.09 1443414_at C78513 EST C78513 2.22 0.00 1450829_at Tnfaip3 tumor necrosis factor, alpha-induced protein 3 2.17 0.00 1427932_s_at 1200016E24Rik RIKEN cDNA 1200016E24 gene 2.16 0.00 1419075_s_at Saa2 serum amyloid A 2 2.10 0.00 1417371_at Peli1 pellino 1 2.01 0.00 1422924_at Tnfsf9 tumor necrosis factor (ligand) superfamily, member 9 2.00 0.00 1454070_a_at Ddhd1 DDHD domain containing 1 1.94 0.01 1425570_at Slamf1 signaling lymphocytic activation molecule family 1.94 0.00 member 1 1417495_x_at Cp ceruloplasmin 1.93 0.14 1419607_at Tnf tumor necrosis factor 1.91 0.00 1420380_at Ccl2 chemokine (C-C motif) ligand 2 1.91 0.01 1418992_at F10 coagulation factor X 1.91 0.10 1418535_at Rgl1 ral guanine nucleotide dissociation stimulator,-like 1 1.89 0.00 1417494_a_at Cp ceruloplasmin 1.86 0.09 1424067_at Icam1 intercellular adhesion molecule 1.86 0.00 1448734_at Cp ceruloplasmin 1.84 0.19 1450698_at Dusp2 dual specificity phosphatase 2 1.84 0.01 1423006_at Pim1 proviral integration site 1 1.82 0.03 1448728_a_at Nfkbiz nuclear factor of kappa light polypeptide gene 1.82 0.02 enhancer in B-cells inhibitor, zeta 1448606_at Edg2 endothelial differentiation, lysophosphatidic acid G- 1.81 0.03 protein-coupled receptor, 2 1448162_at Vcam1 vascular cell adhesion molecule 1 1.79 0.01 1420981_a_at Lmo4 LIM domain only 4 1.76 0.01 1421228_at Ccl7 chemokine (C-C motif) ligand 7 1.72 0.16 1418240_at Gbp2 guanylate nucleotide binding protein 2 1.71 0.07 1419212_at Icosl icos ligand 1.71 0.01 1416649_at Ambp alpha 1 microglobulin/bikunin 1.70 0.00 1419537_at Tcfec transcription factor EC 1.70 0.02 1421578_at Ccl4 chemokine (C-C motif) ligand 4 1.69 0.11 1417143_at Edg2 endothelial differentiation, lysophosphatidic acid G- 1.67 0.04 protein-coupled receptor, 2 1451458_at Tmem2 transmembrane protein 2 1.66 0.06 1425686_at Cflar CASP8 and FADD-like apoptosis regulator 1.66 0.01 1435133_at Ugcg UDP-glucose ceramide glucosyltransferase 1.65 0.00 1421269_at Ugcg UDP-glucose ceramide glucosyltransferase 1.61 0.10 1425412_at Nlrp3 NLR family, pyrin domain containing 3 1.54 0.03 1421487_a_at Nck1 non-catalytic region of tyrosine kinase adaptor protein 1.53 0.01 1 1422474_at Pde4b phosphodiesterase 4B, cAMP specific 1.51 0.00 1418126_at Ccl5 chemokine (C-C motif) ligand 5 1.51 0.58 1435906_x_at Gbp2 guanylate nucleotide binding protein 2 1.50 0.25 1417856_at Relb avian reticuloendotheliosis viral (v-rel) oncogene 1.50 0.01 related B 1417483_at Nfkbiz nuclear factor of kappa light polypeptide gene 1.50 0.11 enhancer in B-cells inhibitor, zeta 1422473_at Pde4b phosphodiesterase 4B, cAMP specific 1.48 0.02 1417496_at Cp ceruloplasmin 1.48 0.50 1423602_at Traf1 Tnf receptor-associated factor 1 1.47 0.02 1460251_at Fas Fas (TNF receptor superfamily member) 1.46 0.01 1449984_at Cxcl2 chemokine (C-X-C motif) ligand 2 1.45 0.01 1450769_s_at Stard5 StAR-related lipid transfer (START) domain containing 1.45 0.01 5 1449591_at Casp4 caspase 4, apoptosis-related cysteine peptidase 1.45 0.14 StAR-related lipid transfer (START) domain containing 1422822_at Stard5 5 1.45 0.01 1416268_at Ets2 E26 avian leukemia oncogene 2, 3' domain 1.44 0.01 1424711_at Tmem2 transmembrane protein 2 1.43 0.05 1420331_at Clec4e C-type lectin domain family 4, member e 1.42 0.02 1421268_at Ugcg UDP-glucose ceramide glucosyltransferase 1.41 0.23 1449317_at Cflar CASP8 and FADD-like apoptosis regulator 1.40 0.09 1448881_at Hp haptoglobin 1.39 0.10 1422526_at Acsl1 acyl-CoA synthetase long-chain family member 1 1.36 0.21 1416576_at Socs3 suppressor of cytokine signaling 3 1.34 0.01 1419354_at Klf7 Kruppel-like factor 7 (ubiquitous) 1.34 0.05 1421457_a_at Samsn1 SAM domain, SH3 domain and nuclear localization 1.31 0.06 signals, 1 1450918_s_at Src Rous sarcoma oncogene 1.30 0.13 1448558_a_at Pla2g4a phospholipase A2, group IVA (cytosolic, calcium- 1.29 0.06 dependent) 1416572_at Mmp14 matrix metallopeptidase 14 (membrane-inserted) 1.29 0.58 1420330_at Clec4e C-type lectin domain family 4, member e 1.28 0.02 1431843_a_at Nfkbie nuclear factor of kappa light polypeptide gene 1.25 0.13 enhancer in B-cells inhibitor, epsilon 1417523_at Plek pleckstrin 1.25 0.13 1449399_a_at Il1b interleukin 1 beta 1.25 0.45 1418641_at Lcp2 lymphocyte cytosolic protein 2 1.23 0.15 1420591_at Gpr84 G protein-coupled receptor 84 1.21 0.44 1450136_at Cd38 CD38 antigen 1.20 0.74 1419355_at Klf7 Kruppel-like factor 7 (ubiquitous) 1.19 0.40 1418401_a_at Dusp16 dual specificity phosphatase 16 1.18 0.11 1450643_s_at Acsl1 acyl-CoA synthetase long-chain family member 1 1.18 0.35 1421065_at Jak2 Janus kinase 2 1.16 0.34 1427381_at Irg1 immunoresponsive gene 1 1.16 0.64 1419254_at Mthfd2 methylenetetrahydrofolate dehydrogenase (NAD+ 1.15 0.73 dependent), methenyltetrahydrofolate cyclohydrolase 1419665_a_at Nupr1 nuclear protein 1 1.14 0.52 1437111_at Zc3h12c zinc finger CCCH type containing 12C 1.12 0.30 1418133_at Bcl3 B-cell leukemia/lymphoma 3 1.11 0.53 1460283_at Mefv Mediterranean fever 1.11 0.59 1419666_x_at Nupr1 nuclear protein 1 1.11 0.60 1418642_at Lcp2 lymphocyte cytosolic protein 2 1.10 0.55 1423240_at Src Rous sarcoma oncogene 1.09 0.72 1449439_at Klf7 Kruppel-like factor 7 (ubiquitous) 1.08 0.50 1450826_a_at Saa3 serum amyloid A 3 1.06 0.90 1433741_at Cd38 CD38 antigen 1.06 0.91 1421066_at Jak2 Janus kinase 2 1.06 0.57 1455899_x_at Socs3 suppressor of cytokine signaling 3 1.05 0.79 1417256_at Mmp13 matrix metallopeptidase 13 1.05 0.90 1448175_at Ehd1 EH-domain containing 1 1.04 0.82 1456212_x_at Socs3 suppressor of cytokine signaling 3 1.04 0.85 1418847_at Arg2 arginase type II 1.04 0.87 1422953_at Fpr-rs2 formyl peptide receptor, related sequence 2 1.02 0.96 1422018_at Hivep2 human immunodeficiency virus type I enhancer binding 1.01 0.93 protein 2 1416012_at Ehd1 EH-domain containing 1 1.01 0.95 1438841_s_at Arg2 arginase type II -1.05 0.85 1416011_x_at Ehd1 EH-domain containing 1 -1.06 0.67 1423017_a_at Il1rn interleukin 1 receptor antagonist -1.10 0.81 1423289_a_at 1810029B16Rik RIKEN cDNA 1810029B16 gene -1.10 0.68 1437917_at D530037H12Rik RIKEN cDNA D530037H12 gene -1.10 0.57 1416010_a_at Ehd1 EH-domain containing 1 -1.12 0.40 1451798_at Il1rn interleukin 1 receptor antagonist -1.14 0.47 1419561_at Ccl3 chemokine (C-C motif) ligand 3 -1.16 0.55 1416701_at Rnd3 Rho family GTPase 3 -1.22 0.57 1449222_at Ebi3 Epstein-Barr virus induced gene 3 -1.23 0.19 1425663_at Il1rn interleukin 1 receptor antagonist -1.23 0.21 1419356_at Klf7 Kruppel-like factor 7 (ubiquitous) -1.25 0.20 1416700_at Rnd3 Rho family GTPase 3 -1.26 0.28 1419684_at Ccl8 chemokine (C-C motif) ligand 8 -1.35 0.54 1422868_s_at Gda guanine deaminase -1.48 0.11 1421720_a_at Dtx2 deltex 2 homolog (Drosophila) -1.51 0.00 1427035_at Slc39a14 solute carrier family 39 (zinc transporter), member 14 -1.55 0.01 1423996_a_at Il4ra interleukin 4 receptor, alpha -1.57 0.03 1450214_at Adora2b adenosine A2b receptor -1.57 0.01 1426600_at Slc2a1 solute carrier family 2 (facilitated glucose transporter), -1.58 0.02 member 1 1437992_x_at Gja1 gap junction membrane channel protein alpha 1 -1.58 0.06 1415800_at Gja1 gap junction membrane channel protein alpha 1 -1.60 0.10 1438650_x_at Gja1 gap junction membrane channel protein alpha 1 -1.65 0.02 1419209_at Cxcl1 chemokine (C-X-C motif) ligand 1 -1.65 0.09 1426599_a_at Slc2a1 solute carrier family 2 (facilitated glucose transporter), -1.71 0.00 member 1 1438945_x_at Gja1 gap junction membrane channel protein alpha 1 -1.73 0.05 1450808_at Fpr1 formyl peptide receptor 1 -1.74 0.21 1419410_at Batf basic leucine zipper transcription factor, ATF-like -1.87 0.00 1425503_at Gcnt2 glucosaminyl (N-acetyl) transferase 2, I-branching -1.89 0.01 enzyme 1428306_at Ddit4 DNA-damage-inducible transcript 4 -2.12 0.03 1423594_a_at Ednrb endothelin receptor type B -2.45 0.00 1428942_at Mt2 metallothionein 2 -2.53 0.06 1416432_at Pfkfb3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase -2.68 0.00 3 1437308_s_at F2r coagulation factor II (thrombin) receptor -2.78 0.01 1452408_at Trex1 three prime repair exonuclease 1 -3.64 0.02 1460197_a_at Steap4 STEAP family member 4 -3.99 0.00 1419082_at Serpinb2 serine (or cysteine) peptidase inhibitor, clade B, -4.23 0.00 member 2 FC refers to expression in Nfkb1-/-Il10-/- BMDM divided by expression in Il10-/- BMDM following stimulation with LPS in the presence of exogenous IL-10. p refers to the P-value between genotypes calculated using the Benjamini-Hochberg correction for multiple comparison testing. All listed genes were induced at least 4 fold by LPS in either Il10-/- or Nfkb1-/-Il10-/- BMDM. Duplicates, ESTs, and hypothetical genes have been removed. Figure S1. Increased IFN-β expression in the absence of Tpl-2/ERK signaling. A) Fold-induction of IFN-β mRNA in WT and Map3k8-/- BMDM 9 hours after stimulation with LPS (10 ng/ml). B) Fold-induction of Ifnb1 mRNA in WT BMDM stimulated for 4h with LPS in the presence or absence of PD184352 (PD).