Emmanuel Derudder

RelB/p50 dimers are differentially regulated by TNF-α and lymphotoxin-β receptor activation: critical roles for p100

Tumor necrosis factor-α (TNF-α) and lymphotoxin-β receptor (LTβR) signaling both play important roles in inflammatory and immune responses through activation of NF-κB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-κB induction in response to TNF-α and LTβR activation. We demonstrate that LTβR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-α induces only RelA/p50 dimers. LTβR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKα but is independent of IKKβ, NEMO/IKKγ, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-α but not LTβ signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-κB pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved.

Immune Surveillance and Therapy of Lymphomas Driven by Epstein-Barr -Virus Protein LMP1 in a Mouse Model

B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.

NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells

BAFF-R-dependent activation of the alternative NF-κB pathway plays an essential role in mature B cell survival. Mutations leading to overexpression of NIK and deletion of the TRAF3 gene are implicated in human multiple myeloma. We show that overexpression of NIK in mouse B lymphocytes amplifies alternative NF-κB activation and peripheral B cell numbers in a BAFF-R-dependent manner, whereas uncoupling NIK from TRAF3-mediated control causes maximal p100 processing and dramatic hyperplasia of BAFF-R-independent B cells. NIK controls alternative NF-κB signaling by increasing the protein levels of its negative regulator TRAF3 in a dose-dependent fashion. This mechanism keeps NIK protein levels below detection even when they cause B cell hyperplasia, so that contributions of NIK to B cell pathologies can easily be overlooked.

Development of immunoglobulin λ-chain–positive B cells, but not editing of immunoglobulin κ-chain, depends on NF-κB signals

By genetically ablating IκB kinase (IKK)-mediated activation of the transcription factor NF-κB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin λ-chain–positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin κ-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-κB signaling. During the first phase, in which NF-κB signaling is dispensable, predominantly κ-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly λ-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding κ-chain. This second phase of development is dependent on NF-κB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

T cell-derived IL-17 mediates epithelial changes in the airway and drives pulmonary neutrophilia.

Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Their production of the cytokine IL-17 is known to induce local recruitment of neutrophils, but the direct impact of IL-17 on the lung epithelium is poorly understood. In this study, we describe a novel mouse model of spontaneous IL-17–driven lung inflammation that exhibits many similarities to asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We used this model to investigate the effects of IL-17 activity on airway epithelium and identified CXCL5 and MIP-2 as important factors in neutrophil recruitment. The neutralization of IL-17 greatly reduces pulmonary neutrophilia, underscoring a key role for IL-17 in promoting chronic airway inflammation. These findings emphasize the role of IL-17 in mediating neutrophil-driven pulmonary inflammation and highlight a new mouse model that may be used for the development of novel therapies targeting Th17 cells in asthma and other chronic pulmonary diseases.

Control of NF-κB Activity by Proteolysis

NF-κB transcription factors are critical regulators of many biological processes such as innate and adaptive immune responses, inflammation, cell proliferation and programmed cell death. This versatility necessitates a highly complex and tightly coordinated control of the signaling pathways leading to their activation. Here, we review the role of proteolysis in the regulation of NF-κB activity, more specifically the contribution of the well-known ubiquitin-proteasome system and the involvement of proteolytic activity of caspases and calpains.

The B-cell antigen receptor integrates adaptive and innate immune signals.

Significance Clonal expansion of antigen-specific B cells during an immune response is necessary for effective antibody production. B cells must integrate signals from their clonally restricted B-cell antigen receptor (BCR) and from nonclonal coreceptors that provide contextual cues to the nature of the antigen. How this is accomplished is unclear. We found that B cells require expression of the BCR for mitogenic responses triggered by coreceptors that recognize innate or T-cell-derived signals. The signaling pathway used by the BCR to license coreceptor-induced proliferation is similar to the previously described BCR-dependent survival signal and, thus, couples B-cell survival and mitogenic responses in an unexpected manner that may have implications for B-cell lymphomagenesis. B cells respond to antigens by engagement of their B-cell antigen receptor (BCR) and of coreceptors through which signals from helper T cells or pathogen-associated molecular patterns are delivered. We show that the proliferative response of B cells to the latter stimuli is controlled by BCR-dependent activation of phosphoinositidyl 3-kinase (PI-3K) signaling. Glycogen synthase kinase 3β and Foxo1 are two PI-3K-regulated targets that play important roles, but to different extents, depending on the specific mitogen. These results suggest a model for integrating signals from the innate and the adaptive immune systems in the control of the B-cell immune response.

IKK phosphorylates RelB to modulate its promoter specificity and promote fibroblast migration downstream of TNF receptors

Significance TNFα induces chemotaxis of inflammatory cells and fibroblasts, but little is known about the signaling mechanisms controlling this action. It is generally accepted that the avian reticuloendotheliosis viral oncogene (v-rel) related B (RelB) NF-κB subunit is not activated downstream of TNF receptors in fibroblasts. Here, we revealed an activating molecular mechanism leading to RelB transcriptional activation that is critical for the control of TNFα-induced fibroblast migration. We show that the IκB kinase (IKK) phosphorylates RelB on serine 472 in response to TNFα, leading RelB to bind to the promoter of critical migration-associated genes, such as the matrix metallopeptidase 3 (MMP3), thereby controlling MMP3 expression and promigration activity. These findings shed light on a crucial regulatory mechanism controlling selective NF-κB target gene expression and cellular response in response to TNFα. TNFα is a potent cytokine that plays a critical role in numerous cellular processes, particularly immune and inflammatory responses, programmed cell death, angiogenesis, and cell migration. Thus, understanding the molecular mechanisms that mediate TNFα-induced cellular responses is a crucial issue. It is generally accepted that global DNA binding activity of the NF-κB avian reticuloendotheliosis viral (v-rel) oncogene related B (RelB) subunit is not induced upon TNFα treatment in fibroblasts, despite its TNFα-induced nuclear accumulation. Here, we demonstrate that RelB plays a critical role in promoting fibroblast migration upon prolonged TNFα treatment. We identified the two kinases IκB kinase α (IKKα) and IκB kinase β (IKKβ) as RelB interacting partners whose activation by TNFα promotes RelB phosphorylation at serine 472. Once phosphorylated on serine 472, nuclear RelB dissociates from its interaction with the inhibitory protein IκBα and binds to the promoter of critical migration-associated genes, such as the matrix metallopeptidase 3 (MMP3). Further, we show that RelB serine 472 phosphorylation status controls MMP3 expression and promigration activity downstream of TNF receptors. Our findings provide new insights into the regulation of RelB activity and reveal a novel link between selective NF-κB target gene expression and cellular response in response to TNFα.

Canonical NF-κB signaling is uniquely required for the long-term persistence of functional mature B cells

Significance Mature B cells are long-lived cells responsible for the Ab production in the immune system. Canonical NF-κB signaling, one of the two discrete pathways activating transcription factors of the NF-κB family, participates in the generation of a normal mature B-cell compartment. However, the role of these signals specifically in mature B cells is still imperfectly defined. Notably, their role in the persistence of follicular B cells, the main mature B-cell subset, is controversial. Here, we show that canonical NF-κB signaling does not contribute to immediate (short-term) survival of follicular B cells, contrasting with the crucial tonic B-cell antigen receptor survival signals, but is required for their long-term persistence as well as functional fitness. Although canonical NF-κB signaling is crucial to generate a normal mature B-cell compartment, its role in the persistence of resting mature B cells is controversial. To resolve this conflict, we ablated NF-κB essential modulator (NEMO) and IκB kinase 2 (IKK2), two essential mediators of the canonical pathway, either early on in B-cell development or specifically in mature B cells. Early ablation severely inhibited the generation of all mature B-cell subsets, but follicular B-cell numbers could be largely rescued by ectopic expression of B-cell lymphoma 2 (Bcl2), despite a persisting block at the transitional stage. Marginal zone (MZ) B and B1 cells were not rescued, indicating a possible role of canonical NF-κB signals beyond the control of cell survival in these subsets. When canonical NF-κB signaling was ablated specifically in mature B cells, the differentiation and/or persistence of MZ B cells was still abrogated, but follicular B-cell numbers were only mildly affected. However, the mutant cells exhibited increased turnover as well as functional deficiencies upon activation, suggesting that canonical NF-κB signals contribute to their long-term persistence and functional fitness.

Igλ+ B cell development but not Igκ editing depends on NF-κB signals

By genetically ablating IκB kinase (IKK)-mediated activation of the transcription factor NF-κB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin λ-chain–positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin κ-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-κB signaling. During the first phase, in which NF-κB signaling is dispensable, predominantly κ-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly λ-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding κ-chain. This second phase of development is dependent on NF-κB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.