Notch-RBP-J Signaling Regulates IRF8 to Promote Inflammatory Macrophage Polarization

RBP-J controls prototypical M1 gene expression

In order to investigate the role of the Notch-RBP-J pathway in macrophage activation, gene expression profiling was performed with wild-type and RBP-J-deficient bone marrow-derived macrophages (BMDMs) stimulated with the TLR4 ligand lipopolysaccharide (LPS), which induces the expression of key M1 proteins like IL-12 and iNOS¹. Efficient deletion of RBP-J in BMDMs from RBP-J-deficient mice (Rbpj^flox/flox, Mx1-Cre) was confirmed at the mRNA and protein expression levels (Supplementary Fig. 1a, b). Microarray analysis showed that approximately 10% of TLR4-inducible genes were partially dependent on RBP-J and that a very broad range of TLR target genes were induced to normal levels in RBP-J-deficient macrophages and thus were RBP-J-independent (Supplementary Fig. 1c and data not shown). However, a small number of LPS-induced genes (< 10) were essentially completely dependent on RBP-J (greater than 80% reduction in RBP-J-deficient cells). Among these genes, Il12a, Il12b and Nos2 dependence on RBP-J expression was confirmed by quantitative PCR (qPCR) (Fig. 1a, b).

To assess the functional and physiological relevance of RBP-J-mediated regulation of M1 genes we examined the in vivo expression of these genes in the myeloid compartment under conditions of inflammation. Upon endotoxin challenge, levels of IL-12p40 protein in serum were significantly reduced in mice with myeloid-specific deletion of Rbpj (Rbpj ^flox/flox, LysM-Cre) compared with wild-type littermate controls (Fig. 1c). iNOS catalyzes production of nitric oxide (NO) in macrophages. In response to LPS stimulation, RBP-J-deficient macrophages produced significantly reduced amounts of NO as measured by levels of the NO metabolite nitrite (Fig. 1d). Because Il12 and Nos2 genes mediate responses against intracellular bacteria, we assessed the role of RBP-J in vivo in host defense against L. monocytogenes, an intracellular pathogen whose successful clearance requires M1 effectors such as IL-12 and iNOS²⁵. Compared with control animals (Rbpj^+/+, LysM-Cre), mice with RBP-J deficiency in the myeloid lineage (Rbpj^flox/flox, LysM-Cre), exhibited enhanced susceptibility to L. monocytogenes infection, as demonstrated by significantly increased bacterial burdens in spleens and livers of the infected animals (Fig. 1e). Taken together, these results show that RBP-J is essential for expression of genes characteristic of the core M1 response in vitro and for manifestation of key myeloid effector functions in vivo.

In addition to promoting M1 gene expression, microarray analysis showed that RBP-J suppressed expression of a group of genes characteristic of M2 macrophage phenotype, confirmed by qPCR (Supplementary Fig. 1d). RBP-J suppressed expression of the key inducer of M2 polarization JMJD3³, indicating that RBP-J plays an inhibitory role in the M2 differentiation program. Although these results suggest that RBP-J might regulate the balance between M1 and M2 polarization, in this study we focused on delineating the mechanisms by which RBP-J regulates the M1 program.

RBP-J-controls M1 gene expression downstream of Notch signaling

RBP-J plays a key role in signal transduction by the canonical Notch pathway. However, Notch-independent RBP-J activities have been reported¹⁴. To assess the role of canonical Notch pathway in RBP-J-mediated regulation of M1 genes, GSI-34, a chemical inhibitor of γ-secretase, was used to abolish signaling from the Notch receptors. GSI-34 treatment of wild-type mouse BMDMs did not have any detectable toxicity effects (data not shown), yet effectively suppressed LPS-induced expression of Il12b (Fig. 2a), suggesting that Il12b induction by LPS requires canonical Notch signaling. Inhibition of γ-secretase by GSI-34 had no effects on the already blunted Il12b expression in RBP-J-deficient macrophages (Fig. 2a), indicating that γ-secretase and RBP-J function in a linear pathway. Another proteolytic event required for Notch signaling activation is the cleavage of receptors by ADAM family proteases, primarily by ADAM10¹⁴. Deficiency of ADAM10 almost completely abolished the induction of RBP-J-dependent genes Il12a, Il12b and Nos2 by LPS in macrophages (Fig. 2b). In contrast, deficiency of another ADAM family protease, ADAM17, did not notably alter LPS-induced expression of RBP-J-dependent M1 genes such as Il12b (Supplementary Fig. 2a and data not shown).

We next asked what Notch receptor(s) are responsible for M1 gene activation. Resting mouse BMDMs express predominantly Notch1 and Notch2 (data not shown). To assess the role for Notch1 in M1 gene expression, we used macrophages from Notch1 heterozygous mice, as complete Notch1 deletion leads to lethality²⁶. Notch1 haploinsufficiency is characterized by approximately 70–80% reduction of Notch1 mRNA expression (Supplementary Fig. 2b)²⁴. Notch1^+/− macrophages exhibited profound defects in induction of RBP-J-dependent M1 genes (Fig. 2c), mimicking the effects of RBP-J deletion (Fig. 1a & 1b), γ-secretase inhibition (Fig. 2a), and ADAM10 deficiency (Fig. 2b). In contrast to Notch1, knocking down expression of Notch2 did not alter LPS-mediated induction of RBP-J-dependent genes such as Il12b (Supplementary Fig. 2c). Knockdown of Notch2 expression in Notch1^+/− cells did not further reduce Il12b expression (Supplementary Fig. 2c), suggesting that Notch2, either alone or in concert with Notch1, does not significantly contribute to the induction of RBP-J-dependent M1 genes. Next, we tested Notch1 function by gain-of-function approaches. Forced expression of NICD1 activated a mouse Il12b promoter-driven reporter construct (Fig. 2d). We also generated mice constitutively expressing NICD1 in myeloid cells (referred to as NICD1^M mice) by crossing LysM-Cre mice with Rosa^Notch mice²⁷. BMDMs from NICD1^M mice were morphologically undistinguishable from wild-type macrophages and expressed mature macrophage markers (Supplementary Fig. 3a–c). NICD1^M BMDMs had increased NICD1 expression and constitutively active Notch signaling, as assessed by expression of the canonical Notch target gene Hes1 (Supplementary Fig. 3d and data not shown). Upon LPS stimulation, NICD1^M macrophages showed augmented induction of M1 genes compared with control macrophages (Fig. 2e). Collectively, these results indicate that Notch signaling components, namely the Notch1-ADAM10-γ-secretase-RBP-J axis, regulate expression of M1 genes.

RBP-J controls IRF8 expression and function

Il12a, Il12b and Nos2, are known to share common mechanisms of regulation, such as dependence on c-Rel^28–30 and dependence on IRF1 and IRF8^6,7,31 and are all categorized as secondary response genes³² (Supplementary Fig. 4). We investigated if the Notch-RBP-J pathway regulates c-Rel, IRF1 or IRF8 expression. Neither c-Rel nor IRF1 expression was significantly altered by RBP-J deficiency (data not shown), suggesting that they are not targets of RBP-J-mediated regulation. It has been reported that IRF8 expression is regulated at the transcriptional level and that induction of IRF8 protein follows mRNA induction and occurs over the course of hours^33,34. In contrast to these previous observations, LPS treatment rapidly (within 15 minutes) and robustly induced IRF8 protein expression in whole cell lysates and nuclear extracts of wild-type BMDMs (Fig. 3a, b, lanes 1–6). The specificity of IRF8 detection by immunoblotting analysis was verified using IRF8-deficient macrophages (Supplementary Fig. 5a). This rapid IRF8 protein induction was validated in various culture conditions and using several protein extraction methods (data not shown). In contrast to the robust LPS-dependent IRF8 induction in wild-type cells, decreased IRF8 expression was observed in whole cell lysates and nuclear extracts from in RBP-J-deficient macrophages (Fig. 3a, b). Expression of other IRF family members such as IRF4 and IRF5 was not affected by RBP-J deficiency (Supplementary Fig. 5b). In a gain-of-function approach, IRF8 protein expression was markedly elevated in macrophages from NICD1^M mice compared with wild-type macrophages (Fig. 3c). These results suggest that Notch-RBP-J is required for the rapid induction of IRF8 protein upon TLR4 stimulation.

Recruitment of IRF8 to its target gene promoters is necessary for binding of RNA polymerase II (Pol II) and subsequent transcriptional activation⁴. Chromatin immunoprecipitation (ChIP) assays showed that activation of wild-type macrophages with LPS led to recruitment of IRF8 to the proximal promoter of Il12b (Fig. 3d). This effect was almost completely abolished in RBP-J-deficient macrophages (Fig. 3d). Recruitment of Pol II to the Il12b promoter was concomitantly decreased in RBP-J-deficient cells (Fig. 3e), suggesting that reduced levels of IRF8 in the absence of RBP-J were not sufficient to assemble the transcription machinery at the Il12b promoter. Overall, our data suggest that RBP-J regulates the expression and transcriptional function of IRF8 downstream of TLR signaling.

We next asked whether decreased IRF8 expression in RBP-J-deficient macrophages explained the low M1 gene expression in these cells. Induction of Il12a, Il12b and Nos2 by LPS was severely decreased in IRF8-deficient macrophages (Fig. 4a). We tested if rescue of IRF8 expression in RBP-J-deficient cells restores Il12a, Il12b and Nos2 expression. Using retroviral transduction, IRF8 expression was restored to approximately wild-type levels in RBP-J-deficient macrophages (Fig. 4b). IRF8 reconstitution nearly completely corrected the defective mRNA expression of Il12b (Fig. 4c) and IL-12p40 protein (Fig. 4d) in RBP-J-deficient cells. IRF8 reconstitution also partially restored expression of Il12a in RBP-J-deficient macrophages (Fig. 4c), while impaired Nos2 expression in RBP-J-deficient cells was not rescued by IRF8 reconstitution (Supplementary Fig. 5c), suggesting involvement of additional factors in RBP-J-regulated Nos2 expression. These results indicate that RBP-J regulates M1 gene expression, at least in part, through IRF8.

RBP-J is required for rapid synthesis of IRF8 protein

Next we investigated the mechanisms by which RBP-J regulates IRF8 protein expression. Because IRF8 expression is known to be regulated at the mRNA level by stimuli such as IFN-γ^34,35, we examined if TLR4 and RBP-J induced Irf8 mRNA accumulation. LPS stimulation for up to 3 hours did not result in notable upregulation of Irf8 mRNA at any of the tested time points (0 to 180 minutes, corresponding to the observed IRF8 protein induction) in wild-type BMDMs (Fig. 5a). As a control, strong induction of Tnf mRNA was observed after LPS treatment (Supplementary Fig. 5d), and IFN-γ pretreatment resulted in induction of Irf8 mRNA in wild-type macrophages as expected (Supplementary Fig. 5e). The above results were also validated by using distinct qPCR primers, which target a different region of Irf8 mRNA³³ (data not shown). These results suggest that rapid induction of IRF8 protein by TLR4 stimulation was not due to increased Irf8 mRNA expression. In addition, RBP-J deficiency did not significantly alter Irf8 mRNA levels at baseline or after LPS treatment (Fig. 5a), suggesting that rapid induction of IRF8 by TLR4 stimulation is regulated at the protein level.

IRF8 is a labile protein³⁶, so we examined whether RBP-J regulates IRF8 protein degradation. The protein synthesis inhibitor cycloheximide (CHX) was added to LPS-stimulated wild-type or RBP-J-deficient macrophages and degradation of IRF8 protein was followed over time. Despite the expected difference in IRF8 protein levels between wild-type and RBP-J-deficient cells prior to CHX treatment, IRF8 protein decreased in a time dependent manner but independent of the RBP-J genotype after CHX treatment (Fig. 5b and Supplementary Fig. 5f). Quantification of IRF8 protein amounts by densitometry revealed that in the absence of new protein synthesis, IRF8 protein decayed at a similar rate in wild-type and RBP-J-deficient cells and that it’s half life was approximately 150 minutes in both cell types (Fig. 5c), a measurement consistent with the estimate from a previous report³⁶. These results suggest that RBP-J does not regulate IRF8 protein degradation. However, addition of CHX to wild-type macrophages prior to LPS stimulation blocked LPS-induced upregulation of IRF8 protein (Fig. 5d, e), suggesting that induction of IRF8 by LPS was the result of new protein synthesis. Metabolic labeling assays showed that LPS stimulation upregulated the incorporation of ³⁵S-methionine/cysteine into newly synthesized IRF8 protein in wild-type macrophages but not in RBP-J-deficient cells (Fig. 5f). These results suggest that rapid IRF8 protein synthesis induced downstream of TLR4 signaling was dependent on RBP-J.

RBP-J controls activation of the MNK1-eIF4E axis

TLR stimulation induces phosphorylation and activation of MNK and subsequent MNK-mediated phosphorylation of eIF4E^37,38, which is required for efficient translation of select protein transcripts³⁹. To investigate the mechanisms by which RBP-J regulates IRF8 protein synthesis we assessed regulation of MNK-eIF4E activity by TLR4 and RBP-J. MNK-eIF4E activity is enhanced by phosphorylation of MNK1 on threonines 197 and 202, and phosphorylation of eIF4E on serine 209¹³. TLR4-induced phosphorylation of MNK1 and eIF4E was greatly diminished in RBP-J-deficient compared to wild-type macrophages (Fig. 6a). This was not due to decreased MNK1 or eIF4E protein expression (Fig. 6a), suggesting that activation of MNK1-eIF4E downstream of TLR4 signaling requires RBP-J.

Activation of MNK1 with subsequent phosphorylation of eIF4E and regulation of translation has been shown to be dependent on Erk and stress-activated MAPKs in various systems^37–39. We examined the role of MAPKs in TLR4-induced activation of MNK1 using pharmacological inhibitors for MEK (U0126), p38 (SB203580) and JNK (SP600125), while an inhibitor of eIF4E phosphorylation by MNK1 (CGP57380)³⁹ served as a positive control (Fig. 6b). While single MAPK inhibitors had modest effects, combination of MEK and p38 inhibitors effectively suppressed TLR4-induced phosphorylation of MNK1 and eIF4E (Fig. 6b), indicating that both Erk and p38 were necessary for activation of the MNK1 pathway by TLR4. RBP-J deficiency did not significantly alter TLR4-induced activation of JNK (Supplementary Fig. 6a), consistent with the idea that JNK is dispensable for MNK1 activation. In contrast, phosphorylation of Erk and MEK (which activates Erk downstream of TLR signaling) was reduced in RBP-J-deficient macrophages (Fig. 6c, Supplementary Fig. 6b). Furthermore, RBPJ deficiency led to decreased phosphorylation of p38 and its upstream kinase MKK3-MKK6 by LPS (Fig. 6d, Supplementary Fig 6b). These results indicate that regulation of TLR4-induced activation of Erk and p38 is one mechanism by which RBP-J controls activation of the MNK1-eIF4E axis. Although the dependence of Erk and p38 signaling on RBP-J was modest, MNK1 activation was dependent on RBP-J, consistent with previous work suggesting a requirement for dual activation of MNK by different MAPKs.

RBP-J targets IRAK2 upstream of MNK-eIF4E

Next, we investigated potential targets of RBP-J upstream of MAPKs and MNK in the TLR4 signaling cascades. IRAK2 is a proximal TLR signaling component that plays a role in the TLR-mediated activation of MNK1 and also functions as a post-transcriptional regulator^38,40. Consistent with previous reports^38,41, acute LPS stimulation of wild-type macrophages did not result in upregulation of IRAK2 expression (Fig. 7a). However, IRAK2 protein expression was greatly diminished in RBP-J-deficient macrophages (Fig. 7a). This effect was specific, as expression of other IRAK family proteins such as IRAK1 was not reduced in RBP-J-deficient macrophages compared with wild-type cells (Supplementary Fig. 6c and data not shown). To determine whether decreased IRAK2 expression contributed to the reduction of M1 gene expression in RBP-J-deficient cells we restored IRAK2 expression in RBP-J-deficient macrophages by retroviral transduction. IRAK2 reconstitution partially corrected the phenotype (Fig. 7b), suggesting that the requirement of RBP-J in M1 gene induction is, at least in part, due to it’s regulation of IRAK2.

Next we investigated the mechanisms by which RBP-J signaling regulates IRAK2 expression. Levels of Irak2 mRNA did not significantly differ between wild-type and RBP-J-deficient cells at baseline or after LPS stimulation (Fig. 7c), indicating that RBP-J does not regulate Irak2 gene expression. We also tested if RBP-J deficiency decreases IRAK2 protein synthesis and/or increases IRAK2 protein degradation. We assessed IRAK2 protein synthesis by metabolic labeling assays and found that RBP-J deficiency resulted in attenuated synthesis of IRAK2 protein as evidenced by reduced incorporation of ³⁵S-methionine/cysteine at multiple labeling time points (Fig. 7d). In addition, in the presence of the protein synthesis inhibitor CHX, IRAK2 degraded at a faster rate in RBP-J-deficient macrophages than in control macrophages under LPS-stimulated conditions (Fig. 7e). Therefore, both decreased synthesis and increased degradation contributed to the low IRAK2 protein levels observed in RBP-J-deficient cells. We also examined IRAK2 protein expression in NICD1^M macrophages and found increased IRAK2 protein expression compared to macrophages from wild-type mice (Fig. 7f). Increased IRAK2 protein expression in NICD1^M cells was not due to augmented Irak2 mRNA expression (Supplementary Fig. 6d), supporting the notion that Notch-RBP-J signaling regulates IRAK2 expression post-transcriptionally.

To test the role of IRAK2 in mediating RBP-J-dependent TLR4-induced signaling events we evaluated activation of MAPKKs and MAPKs in cells in which IRAK2 expression was knocked down using RNAi. Diminished IRAK2 expression (Supplementary Fig. 6e) resulted in impaired activation of the MEK-Erk as well as MKK3-MKK6-p38 pathways upon LPS stimulation (Supplementary Fig. 6f), but did not affect JNK phosphorylation (Supplementary Fig. 6g). Overall, these results demonstrate that the Notch-RBP-J pathway controls a TLR4- activated MAPK-MNK-eIF4E signaling cascade by regulating expression of IRAK2 protein.

MNK1-eIF4E controls TLR4-induced IRF8 protein synthesis

We sought to link RBP-J-mediated regulation of MAPK-MNK1-eIF4E signaling with regulation of IRF8 protein induction. In LPS-activated macrophages, IRF8 expression was strongly decreased when both Erk and p38 activation were pharmacologically inhibited using U0126 and SB203580 respectively, whereas inhibition of JNK using SP600125 did not have discernible effects on IRF8 protein amounts (Fig. 8a). Combined inhibition of Erk and p38 almost completely abolished induction of M1 genes (Fig. 8b) and IL-12 protein (Supplementary Fig. 7a) by LPS, suggesting that both Erk and p38 were necessary for IRF8 protein expression and subsequent induction of M1 genes in TLR4-stimulated macrophages. Treatment of macrophages with the MNK inhibitor CGP57380 suppressed LPS-induced eIF4E phosphorylation and IRF8 protein expression in a dose-dependent manner (Fig. 8c). RNAi knockdown of MNK1 in macrophages led to decreased eIF4E phosphorylation and resulted in attenuated induction of IRF8 by LPS (Fig. 8d, Supplementary Fig. 7b). However, it did not affect expression of Irf8 mRNA (Supplementary Fig. 7c). Transduction of macrophages with a retrovirus expressing a dominant negative mutant of MNK1 that lacks kinase activity, and thus is unable to phosphorylate eIF4E⁴², blunted LPS-activated IRF8 protein induction (Fig. 8e). Inhibition of MNK activity by CGP57380 suppressed TLR4-induced expression of Il12a, Il12b and Nos2 (Fig. 8f), without apparent toxicity (data not shown) and global interference with TLR responsiveness (Supplementary Fig. 7d). Altogether, these experiments support a role for MNK-eIF4E in the TLR4-induced expression of IRF8 protein and induction of IRF8 target genes. A model of the regulation of M1 macrophage polarization through Notch-TLR signaling crosstalk is shown in Supplementary Fig. 8.

Cells and reagents

Murine BMDMs were obtained as described¹⁷ and maintained in DMEM supplemented with 10% FBS and 10% L929 cell supernatant as conditioned medium providing macrophage colony stimulating factor (M-CSF). Recombinant mouse IFN-γ was from Peprotech and used at 10 ng/ml. Cell culture grade LPS and CHX were purchased from Sigma-Aldrich. Pam3Cys was purchased from EMC Microcollections. LPS was used as 1 ng/ml and Pam3Cys was used at 10 ng/ml unless otherwise noted. A γ-secretase inhibitor GSI-34 was used at 10 µM as previously described⁴⁶. U0126, SB203580, SP600125, and CGP57380 were from Calbiochem.

Mice

The experiments using mice were approved by Institutional Animal Care and Use Committees at the Hospital for Special Surgery, Columbia University, and Christian Albrechts Universität Kiel. C57/BL6 mice were purchased from Jackson Laboratory. Rbpj^flox/flox mice were kindly provided by Tasuku Honjo. Mice with a myeloid-specific deletion of Rbpj (Rbpj^flox/flox, LysM-Cre) were as described previously¹⁷ and used for in vivo experiments given the tissue specificity of gene deletion. Mice with an inducible deletion of Rbpj (Rbpj^flox/flox, Mx1-Cre) were generated by crossing Rbpj^flox/flox animals to animals with an Mx1 promoter driven Cre transgene on the C57/BL6 background (Jackson Laboratory). Littermates with Rbpj^flox/flox, Mx1-Cre (RBPJ-KO) or Rbpj^+/+, Mx1-Cre (wild-type; WT) genotypes were intraperitonially injected with 200 µg/mouse of PolyI:C three times in five days to induce deletion and mice were used for experiments two weeks later. For all in vitro experiments involving RBP-J-deficient macrophages, cells were derived from Rbpj^flox/flox, Mx1-Cre animals where consistent and strong deletion of Rbpj (approximately 80%) was observed and the conditional deletion was controlled for expression of Cre and genetic background. Adam10^flox/flox animals were as described⁴⁷ and Adam10^flox/flox, Mx1-Cre mice were generated and used in a manner similar to that of Rbpj^flox/flox, Mx1-Cre animals. Adam17^flox/flox, LysM-Cre animals were generated by crossing Adam17^flox/flox animals⁴⁸ to LysM-Cre mice on the C57/BL6 background (Jackson Laboratory). Notch1^+/− mice were kindly provided by Thomas Gridley²⁶. Rosa^Notch mice in which the mouse cDNA encoding constitutively active NICD1 was knocked into the ubiquitously expressed Rosa26 locus, followed by internal ribosome entry sequence-EGFP, and preceded by a STOP fragment flanked by loxP sites²⁷ were obtained from the Jackson Laboratory. We generated mice with myeloid-specific constitutive expression of NICD1 by crossing Rosa^Notch mice with LysM-Cre mice (referred to as NICD1^M mice). Gender and age matched LysM-Cre mice were used as controls. NICD1^M macrophages were obtained by culturing bone marrow cells from NICD1^M mice for 5 days in M-CSF-containing conditioned medium. At the end of the culture period, adherent cells were collected and replated for experiments. Irf8^−/− mice were as described⁵. All experiments involving knockout mice were performed with sex-matched littermates of desired genotypes as controls. Bone marrow chimeras were generated as previously described⁴⁹. Briefly, recipient C57BL/6 mice were subjected to irradiation at a dose of 1000 cGy, followed by intravenous injection of 1 × 10⁶ donor bone marrow cells from the myeloid-specific RBP-J-deficient mice with the genotype Rbpj^flox/flox, LysM-Cre or the WT littermate controls with the genotype Rbpj^+/+, LysM-Cre. Chimeric mice were used for experiments 6 weeks after the initial bone marrow transfer.

mRNA isolation and quantitative PCR (qPCR)

RNA was extracted from whole cell lysates with an RNeasy Mini kit (Qiagen) and was reverse transcribed with a First Strand cDNA synthesis kit (Fermentas). qPCR was performed in triplicate wells with an iCycler IQ thermal cycler and detection system (Biorad) using gene-specific primers. Threshold cycle numbers were normalized to triplicate samples amplified with primers specific for glyceraldehyde-3-phosphate dehydrogenase (Gapdh).

Enzyme-linked immunosorbent assay (ELISA)

Cytokine secretion was quantified by ELISA kits from BD Pharmaingen and nitric oxide production was measured using Greiss reagent (Sigma) according to manufacturers’ instructions.

Listeria monocytogenes infection

Mice were infected intravenously with 3 × 10³ L. monocytogenes strain 10403S, as described previously⁴⁹. At day 3.5 post infection, spleens and livers were harvested and dissociated in PBS containing 0.05% Triton X-100, and bacterial colony-forming units (CFUs) were determined by plating on brain-heart infusion agar plates.

Immunoblot analysis

Whole cell lysates were prepared by direct lysis in SDS loading buffer. For immunoblot analysis, lysates were separated by 10% SDS-PAGE and transferred to a PVDF membrane for probing with antibody. Polyclonal antibodies against IRF8, p38, c-Rel, TBP and SHP2 were from Santa Cruz Biotechnology. Antibodies against IRAK2 and β-tubulin were from Abcam. Anibodies against p-MEK1-MEK2 (Ser217/221), p-MKK3-MKK6 (Ser189/207), p-JNK (Thr183/Tyr185), p-Erk (Thr202/Tyr204), p-p38 (Thr180/Tyr182), p-MNK1 (Thr197/202), p-eIF4E (Ser209), MNK1, eIF4E, IκBα, IRF4, IRF5, Erk1-Erk2, and NICD1 were from Cell signaling. An antibody against NICD2 was from University of Iowa Developmental Studies Hybridoma Bank. Anti-RBP-J rabbit serum was a generous gift from Elliott Kieff and Jon C. Aster⁵⁰.

Transient transfection and luciferase assay

Murine Il12b promoter reporter plasmid containing sequences from positions −356 to +55 was kindly provided by Stephen T. Smale. RAW264.7 cells were cotransfected in duplicates with the Il12b reporter plasmid and an expression vector encoding NICD1, a gift from Raphael Kopan using Lipofectamine LTX reagent (Invitrogen). pRL-TK plasmid encoding Renilla luciferase (Promega) was used as an internal control. Dual-Luciferase Reporter Assay System (Promega) was utilized to detect the luciferase activity of cell lysates 36 hours after transfection.

RNA interference (RNAi)

Prevalidated specific short interfering RNAs (siRNA) targeting murine Notch2, IRAK2, MNK1, and non-targeting control siRNA were purchased from Dharmacon. siRNAs were transfected into mouse BMDMs using the TransIT TKO transfection reagent (Mirus Bio) according to the manufacturers’ instructions.

Chromatin immunoprecipitation (ChIP)

This assay was performed as previously described¹⁷ with slight modifications. In brief, 8–10 × 10⁶ cells were crosslinked with 0.75 % formaldehyde. After being lysed in 8 ml of lysis buffer, the pellets were re-suspended in cold RIPA buffer and sonicated on ice at power setting 5 in 20 s bursts of 6 cycles. Lysates were then cleared by centrifugation, and incubated with 2 µg of goat anti-IRF8 (C-19, Santa Cruz Biotechnology) or of monoclonal anti-RNA polymerase II antibody (Millipore) overnight with rotation. Same amounts of normal goat IgG or normal mouse IgG were used as controls, respectively. Antibody incubation was followed by incubation with 45 µl of 33% Protein A/G agarose slurry (Santa Cruz Biotechnology) for 1.5 hours at 4°C. Then the agarose slurry bond complexes were digested with proteinase K and phenol: chloroform was used to purify DNA for qPCR. The unrelated 28S rRNA was used for qPCR normalization. The value of IgG control in untreated cells was set to 1. Primers used for qPCR were as follows: Il12b locus forward: 5’-CACACTGGACCAAAGGGACT-3’; Il12b locus reverse: 5’-CTTTGCTTCCCTAGCACCT-3’; 28S rRNA forward: 5’-GATCCTTCGATGTCGGCTCTTCCTATC-3’; 28S rRNA reverse: 5’-AGGGTAAAACTAACCTGTCTCACG-3’.

Metabolic labeling

8–10 × 10⁶ of BMDMs cultured in complete medium were starved for 30 minutes in methonine and cysteine-free DMEM supplemented with 5% dialysed FBS. Then the cells were labeled with ³⁵S-Methionine/cysteine Labeling Mix (Perkin Elmer) at a final concentration of 100 µCi/ml. At the end of the labeling period, cells were washed twice with cold PBS and lysed in 500 µl of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM EGTA, 0.2 mM EDTA, 1% Nonidet P-40, 1 mM dithiothreitol) supplemented with protease inhibitors (Roche). Immunoprecipitation of IRF8 and IRAK2 was performed using a goat polyclonal anti-IRF8 antibody (C-19, Santa Cruz Biotechnology) and a rabbit polyclonal anti-IRAK2 antibody (Abcam) respectively. Immunoprecipitates were resuspended in 20 µl of SDS-PAGE loading buffer for subsequent electrophoresis on a 7.5% SDS-PAGE. The gel was dried and placed on a film for autoradiography overnight at −80°C.

Retroviral transduction

Plat-E cells seeded at a density of 2 × 10⁶ per well into 6-well plates were cultured overnight and then transfected using Fugene HD (Roche) with 3 µg of the retroviral vector. After 48 hours, the viral supernatant was collected and centrifuged at 1500 rpm. 1.5 ml of viral supernatant was used to transduce 4 × 10⁵ BMDMs in the presence of 8 µg/ml of polybrene (Sigma). BMDMs were used for assays 48 hours after viral transduction. For pMx-Puro vector-based transduction, virally-transduced macrophages were selected in the puromycin-containing medium for 4 days prior to being replated for assays. pMX-Irf8-IRES-EGFP retroviral vector and pMX-IRES-EGFP control vector were kindly provided by Masamichi Takami. A retroviral construct encoding IRAK2 and an empty-vector control construct were generous gifts from Shizuo Akira⁴¹. WT and DN (Thr197Ala-Thr202Ala mutant) MNK1 expression constructs were kindly provided by Jonathan A. Cooper⁴² and cDNA fragments encoding WT and DN MNK1 were subcloned into the pMx-Puro retroviral vector.

Flow cytometry

BMDMs were harvested after 5 days of culture in M-CSF-containing conditioned medium and stained with an APC-conjugated anti-CD11b antibody (BD Pharmingen) and a PE-conjugated anti-F4/80 antibody (eBioscience). Cells were washed three times and analyzed on a FACScan flow cytometer (BD) using CellQuest software (BD).