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Control of Relb During Dendritic Cell Activation

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Control of Relb During Dendritic Cell Activation ARTICLES Control of RelB during dendritic cell activation integrates canonical and noncanonical NF-kB pathways Vincent F-S Shih1, Jeremy Davis-Turak1, Monica Macal2, Jenny Q Huang1, Julia Ponomarenko3, Jeffrey D Kearns1,5, Tony Yu1, Riku Fagerlund1, Masataka Asagiri1,4, Elina I Zuniga2 & Alexander Hoffmann1 The NF-kB protein RelB controls dendritic cell (DC) maturation and may be targeted therapeutically to manipulate T cell responses in disease. Here we report that RelB promoted DC activation not as the expected RelB-p52 effector of the noncanonical NF-kB pathway, but as a RelB-p50 dimer regulated by canonical IkBs, IkBa and IkB«. IkB control of RelB minimized spontaneous maturation but enabled rapid pathogen-responsive maturation. Computational modeling of the NF-kB signaling module identified control points of this unexpected cell type–specific regulation. Fibroblasts that we engineered accordingly showed DC-like RelB control. Canonical pathway control of RelB regulated pathogen-responsive gene expression programs. This work illustrates the potential utility of systems analyses in guiding the development of combination therapeutics for modulating DC-dependent T cell responses. DCs are specialized sentinel immune cells essential in both innate and rejection of allogenic organ transplants by determining the balance adaptive immunity. DC progenitors differentiate to become imma- of associated activated or regulatory T cells7. These insights have ture DCs that populate both nonlymphoid and lymphoid tissues and prompted investigations of cell-based therapies for autoimmune perform immune-surveillance functions. When encountering patho- diseases using RelB-silenced DCs11. gens or pathogen-associated molecular patterns (PAMPs), immature Despite the potential clinical importance of RelB, the molecular DCs undergo a maturation program that determines their role in the mechanisms that control its activity in DCs have remained unclear. adaptive immune response1. A hallmark of DC maturation is expres- Mouse embryonic fibroblasts (MEFs) have served as a useful model sion of major histocompatibility complex molecules (MHC), T cell system for many signaling studies. Detailed biochemical studies in co-stimulatory molecules (CD40, CD80 or CD86) and cytokines MEFs have shown that unlike classical NF-κB (the RelA-p50 dimer), (for example, interleukin 23; IL-23) in addition to a gene expression RelB is not activated from a latent cytoplasmic pool via the NEMO- © All rights reserved. 2012 Inc. Nature America, program of intracellular factors that enable effective antigen uptake, dependent, ‘canonical’ signaling pathway but via the ‘noncanonical’ processing and presentation, and T cell activation. In addition, pro- NF-κB pathway that involves proteolysis and processing of newly duction of inflammatory molecules such as nitric oxide and cytokines synthesized NF-κB2 p10012–14. Consistent with the critical role of such as tumor necrosis factor (TNF) and interferon underlies DC RelB in DCs, noncanonical signaling pathway components such as functions in innate immune responses2,3. DCs have thus attracted the signaling protein NIK and Nfkb2 gene have been reported to be attention for engineering or modulating immune-based therapies4. required for proper DC functions10,15. However, RelB has also been The transcription factor NF-κB protein RelB is highly expressed found to be rapidly activated in DCs by canonical pathway stimuli in antigen-presenting cells5 and is critical for DC maturation, DC TNF and lipopolysaccharide (LPS)16–19, and the canonical signaling function as antigen-presenting cells6 and DC-mediated immunity. pathway component TRAF6 has been shown to be essential9. These Specifically, small interfering RNA–mediated silencing of RelB expres- reports suggest that the mechanism by which RelB activity is control- sion radically alters the DC maturation process and results in blunted led in DCs may be different than what has been described in MEFs. In antigen-specific T cell responses in vitro and in vivo7. RelB-deficient DCs, the molecular control mechanisms must provide for constitutive mice have deficiencies in splenic DC subsets8,9 but other critical roles RelB expression to enable rapid and decisive induction of matura- of RelB in DCs may be masked by other cell types in which RelB- tion programs after exposure to pathogens or PAMPs but must limit ­deficiency leads to functionally opposite phenotype: notably, T cells spontaneous maturation of DCs in their absence. are hyperactive in these null mice, whereas DC-specific deletion of In this study, we elucidated the molecular mechanisms responsi- the RelB-controlling kinase NIK results in deficient T cell responses10. ble for regulating RelB in DCs. We used a systems biology approach Indeed, the extent of RelB activation determines the tolerance or of iterative computational modeling and quantitative experimental 1Signaling Systems Laboratory, Department of Chemistry and Biochemistry and San Diego Center for Systems Biology, University of California, San Diego, La Jolla, California, USA. 2Division of Biological Sciences, University of California, San Diego, La Jolla, California, USA. 3San Diego Supercomputer Center, University of California, San Diego, La Jolla, California, USA. 4Innovation Center for Immunoregulation and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyoku, Kyoto, Japan. 5Present address: Merrimack Pharmaceuticals, Cambridge, Massachusetts, USA. Correspondence should be addressed to A.H. ([email protected]). Received 28 November 2011; accepted 29 August 2012; published online 21 October 2012; doi:10.1038/ni.2446 NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1 ARTICLES a b c TNF or LPS LTβ 4 8 LPS LPS 100 100 3 6 2 4 NEMO NIK mRNA IKK IKK 50 50 (fold) 1 (fold) 2 Rela 0 RelA protein 0 nRelA (nM) 50 52 p100 Ø nRelB (nM) A B 0 1 2 3 0 1 2 3 4 8 Time (h) Time (h) 3 6 Canonical 2 4 mRNA LT LT (fold) (fold) Noncanonical β β 1 2 Ø α β ε 100 100 Relb Cytoplasm 0 RelB protein 0 50 50 8 Nucleus 4 8 3 6 6 nRelB (nM) nRelA (nM) 0 8 16 24 0 8 16 24 mRNA 2 4 4 (fold) Time (h) Time (h) (fold) 1 (fold) 2 2 Inflammation Organogenesis p52 protein p100 protein Nfkb2 0 0 0 MEF MEF MEF BMDCBMDM BMDCBMDM BMDCBMDM Figure 1 A MEF-based kinetic model does not account for d e Cytoplasm f Cytoplasm g RelB regulation in DCs. (a) Schematic of the distinct Day: 1 3 6 10 40 Nucleus Nucleus canonical and noncanonical NF- B pathways identified in B MEF BMDCBMDM κ IKK1 30 120 MEFs12. Inflammatory signals lead to activation of the p100 p100 ) 90 20 3 NEMO-containing kinase complex that triggers IκBα, IκBβ 60 RelB 10 RelB (nM) 10 and IκBε (α, β and ε), degradation and the release of RelA-p50 p52 × 30 Computational RelA into the nucleus. Developmental signals activate NIK-IKK1 kinase β-actin 0 0 MEFDC Experimental Rel -actin complex that results in p100 processing, which allows for DC β (per nucleus/cytoplasm RelB-p52 nuclear translocation. (The IκBδ pathway is not shown for sake of clarity21). A, RelA; B, RelB; 50, p50; 52, p52; and Ø, sink (from which proteins are synthesized and into which they are degraded). (b) Computational simulations using the MEF-based kinetic model version 5.0–MEF (Supplementary Note) of nuclear RelA or RelB activity (nRelA and nRelB, respectively) induced by LPS or LTβ stimulation. (c) Quantification of Rela, Relb and Nfkb2 transcripts by quantitative RT-PCR (left) and of RelA, RelB, p100 and p52 proteins by immunoblot (right); numbers per cell in resting MEFs, BMDMs and BMDCs, graphed relative to the respective value in MEFs. (d) IKK1 and p52 abundance increase during DC differentiation. Whole-cell extracts prepared from BMDC culture during a differentiation time course (days 1–10) were subjected to IKK1, p100 and p52 immunoblotting. β-actin served as a loading control. (e) In silico simulation of RelB cellular distribution using the mathematical model version 5.0–MEF describing NF-κB activation in MEFs as in b or in model version 5.0–DC incorporating DC-specific parameters derived from c,d (Supplementary Note). (f) Quantification of RelB molecules per wild-type (WT) BMDC distributed in cytoplasmic and nuclear fraction. Quantification methods are described in Supplementary Figure 1. (g) RelB, RelA and p100 immunoblots of cytoplasmic extracts prepared from the indicated cell types. Data are representative of at least three independent experiments (error bars, s.d.; n = 3). analyses of the NF-κB signaling network in DCs to reveal that RelB To adapt the model to DCs, we first measured the expression activity was limited by classical IκBs, IκBα and IκBε, and regulated via of key NF-κB proteins in bone marrow–derived DCs (BMDCs) the canonical pathway. Modeling studies identified two DC-specific in comparison to that in MEFs and bone marrow–derived macro- control points that render RelB subject to regulation by the canonical phages (BMDMs). Relative to the expression of the housekeeping pathway, and we demonstrated their sufficiency by engineering MEFs gene β-actin (Actb), expression of Rela mRNA was similar in BMDCs, accordingly to produce DC-like RelB control. Finally, gene expression BMDMs and MEFs, and the relative amount of RelA protein in these © All rights reserved. 2012 Inc. Nature America, profiling revealed that RelB-dependent gene expression programs cell types correlated (Fig. 1c). In contrast, we observed threefold to regulated by the canonical pathway activity control DC-orchestrated sixfold more Relb mRNA and protein expression in BMDCs than immune responses. MEFs and BMDMs (Fig. 1c and Supplementary Fig. 1a). p100, encoded by the Nfkb2 gene, is known to inhibit RelB. We there- RESULTS fore tested whether p100 expression correlated with enhanced RelB Developing a DC-specific model for NFkB signaling expression in BMDCs. We observed 3.5-fold more Nfkb2 mRNA in The established view of NF-κB signaling comprises two separate BMDCs, but quantitative immunoblotting showed little difference in pathways (Fig.
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