Nicole Heise

The diverse roles of IRF4 in late germinal center B‐cell differentiation

Summary:  Interferon regulatory factor 4 (IRF4) is a member of the IRF family of transcription factors and is expressed in most cell types of the immune system. Within the B‐cell lineage, IRF4 is expressed in all developmental stages except during the germinal center (GC) reaction. IRF4 expression, however, is upregulated during exit from the GC reaction and has been demonstrated to have critical functions in at least three key developmental processes: the termination of the GC B‐cell transcriptional program, immunoglobulin (Ig) class switch recombination (CSR), and plasma cell development. Herein, we attempt to reconcile the often contradictory findings regarding IRF4 into a model to explain the role of IRF4 in the transcription factor networks that operate within exiting GC B cells. In addition, a deregulation of the biological programs controlled by IRF4 has recently been implicated in the pathogenesis of various B‐cell–derived malignancies. Determining the specific functions of IRF4 in the markedly diverse developmental processes that coordinate B‐cell development is therefore likely to have important implications for understanding these malignancies and devising therapeutic interventions.

Germinal center B cell maintenance and differentiation are controlled by distinct NF-κB transcription factor subunits

Heise et al. find that the NF-κB subunits c-REL and RELA in B cells play distinct roles during the germinal center reaction. While RELA stimulates the emergence of plasma cells from the germinal center, c-REL supports maintenance of the reaction over time, possibly by inducing a metabolic gene program connected to cell proliferation.

NEMO Prevents Steatohepatitis and Hepatocellular Carcinoma by Inhibiting RIPK1 Kinase Activity-Mediated Hepatocyte Apoptosis

Summary IκB kinase/nuclear factor κB (IKK/NF-κB) signaling exhibits important yet opposing functions in hepatocarcinogenesis. Mice lacking NEMO in liver parenchymal cells (LPC) spontaneously develop steatohepatitis and hepatocellular carcinoma (HCC) suggesting that NF-κB prevents liver disease and cancer. Here, we show that complete NF-κB inhibition by combined LPC-specific ablation of RelA, c-Rel, and RelB did not phenocopy NEMO deficiency, but constitutively active IKK2-mediated NF-κB activation prevented hepatocellular damage and HCC in NEMOLPC-KO mice. Knock-in expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) prevented hepatocyte apoptosis and HCC, while RIPK1 ablation induced TNFR1-associated death domain protein (TRADD)-dependent hepatocyte apoptosis and liver tumors in NEMOLPC-KO mice, revealing distinct kinase-dependent and scaffolding functions of RIPK1. Collectively, these results show that NEMO prevents hepatocarcinogenesis by inhibiting RIPK1 kinase activity-driven hepatocyte apoptosis through NF-κB-dependent and -independent functions.

IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression and activity

The transcription factor IRF4 limits the retention of B cells in the marginal zone by inhibiting NOTCH2 signaling.

Transcription factors of the alternative NF-κB pathway are required for germinal center B-cell development

Significance In many human B-cell cancers, a complex signaling cascade called NF-κB is abnormally activated by genetic mutations. The uncontrolled activity of NF-κB because of genetic mutations promotes the formation of B-cell tumors. The NF-κB cascade is comprised of two distinct pathways. We here define the role of one of these routes, called the alternative NF-κB pathway, in the normal cells from which these B-cell tumors are derived, namely germinal center (GC) B cells or plasma cells (PCs). We found that the inactivation of the alternative NF-κB pathway led to the loss of GC B cells and impaired PC development. Understanding the role of this pathway in normal cells may provide important insights into how aberrant activation promotes B-cell tumors. The NF-κB signaling cascade relays external signals essential for B-cell growth and survival. This cascade is frequently hijacked by cancers that arise from the malignant transformation of germinal center (GC) B cells, underscoring the importance of deciphering the function of NF-κB in these cells. The NF-κB signaling cascade is comprised of two branches, the canonical and alternative NF-κB pathways, mediated by distinct transcription factors. The expression and function of the transcription factors of the alternative pathway, RELB and NF-κB2, in late B-cell development is incompletely understood. Using conditional deletion of relb and nfkb2 in GC B cells, we here report that ablation of both RELB and NF-κB2, but not of the single transcription factors, resulted in the collapse of established GCs. RELB/NF-κB2 deficiency in GC B cells was associated with impaired cell-cycle entry and reduced expression of the cell-surface receptor inducible T-cell costimulator ligand that promotes optimal interactions between B and T cells. Analysis of human tonsillar tissue revealed that plasma cells and their precursors in the GC expressed high levels of NF-κB2 relative to surrounding lymphocytes. Accordingly, deletion of nfkb2 in murine GC B cells resulted in a dramatic reduction of antigen-specific antibody-secreting cells, whereas deletion of relb had no effect. These results demonstrate that the transcription factors of the alternative NF-κB pathway control distinct stages of late B-cell development, which may have implications for B-cell malignancies that aberrantly activate this pathway.

Cutting Edge: NF-κB p65 and c-Rel Control Epidermal Development and Immune Homeostasis in the Skin

Psoriasis is an inflammatory skin disease in which activated immune cells and the proinflammatory cytokine TNF are well-known mediators of pathogenesis. The transcription factor NF-κB is a key regulator of TNF production and TNF-induced proinflammatory gene expression, and both the psoriatic transcriptome and genetic susceptibility further implicate NF-κB in psoriasis etiopathology. However, the role of NF-κB in psoriasis remains controversial. We analyzed the function of canonical NF-κB in the epidermis using CRE-mediated deletion of p65 and c-Rel in keratinocytes. In contrast to animals lacking p65 or c-Rel alone, mice lacking both subunits developed severe dermatitis after birth. Consistent with its partial histological similarity to human psoriasis, this condition could be prevented by anti-TNF treatment. Moreover, regulatory T cells in lesional skin played an important role in disease remission. Our results demonstrate that canonical NF-κB in keratinocytes is essential for the maintenance of skin immune homeostasis and is protective against spontaneous dermatitis.

Unexpected functions of nuclear factor-κb during germinal center B-cell development: implications for lymphomagenesis.

Purpose of reviewB-cell tumors originating from the transformation of germinal center B cells frequently harbor genetic mutations, leading to constitutive activation of the nuclear factor-&kgr;B (NF-&kgr;B) signaling pathway. The present review highlights recent insights into the roles of separate NF-&kgr;B transcription factors in germinal center B-cell development and discusses implications of the results for germinal center lymphomagenesis. Recent findingsUnderstanding how aberrant NF-&kgr;B activation promotes tumorigenesis requires the understanding of the role of NF-&kgr;B in the tumor-precursor cells. Despite extensive knowledge on NF-&kgr;B biology, the function of this complex signaling pathway in the differentiation of germinal center B cells is largely unknown. The present review will discuss recent findings that revealed distinct roles of separate NF-&kgr;B transcription factors during the germinal center reaction in the context of germinal center lymphomagenesis. Most notably, a single NF-&kgr;B subunit, c-REL, was found to be required for the maintenance of the germinal center reaction and was associated with the activation of a metabolic program that promotes cell growth. SummaryIdentifying the biological roles of the separate NF-&kgr;B transcription factor subunits in germinal center biology will help to better understand the pathogenic consequences of their constitutive activation in B-cell tumors. This knowledge may be exploited for the development of targeted antitumor therapies aimed at inhibiting selectively those components of aberrant NF-&kgr;B activity which contribute to pathogenesis.

Somatic Hypermutation and Affinity Maturation Analysis Using the 4-Hydroxy-3-Nitrophenyl-Acetyl (NP) System

Somatic hypermutation of immunoglobulin variable region (IgV) genes and affinity maturation of the antibody response are the hallmarks of the germinal center (GC) reaction in T cell-dependent immune responses. Determining the consequences of the experimental manipulation of the GC response on somatic hypermutation and affinity maturation requires the availability of a system that allows measuring these parameters. Immunization of mice of the C57/Bl6 genetic background with the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein leads to the predominant usage of one particular IgV heavy chain gene segment, V186.2, among the responding B cells. Moreover, a specific somatic mutation in codon 33 of V186.2 that leads to a tryptophan to leucine amino acid exchange increases the affinity of the corresponding antibody by ~10-fold, thus representing a molecular marker for affinity maturation. In addition, due to the simplicity of the antigen and the virtual absence of NP-specific plasma cells prior to immunization, NP-based immunizations represent ideal tools to quantify the plasma cell response by measuring NP-specific antisera by ELISA and the generation of NP-specific plasma cells by ELISPOT analysis. We here describe approaches to (1) measure the anti-NP plasma cell response by ELISA and ELISPOT analysis, and to (2) amplify and sequence V186.2 rearrangements from GC B cells and plasma cells to determine the level of somatic hypermutation and the extent of affinity maturation in the anti-NP response.

Differential requirements for the canonical NF-κB transcription factors c-REL and RELA during the generation and activation of mature B cells.

Signaling through the canonical nuclear factor‐κB (NF‐κB) pathway is critical for the generation and maintenance of mature B cells and for antigen‐dependent B‐cell activation. c‐REL (rel) and RELA (rela) are the downstream transcriptional activators of the canonical NF‐κB pathway. Studies of B cells derived from constitutional rel knockout mice and chimeric mice repopulated with rela–/– fetal liver cells provided evidence that the subunits can have distinct roles during B‐cell development. However, the B cell‐intrinsic functions of c‐REL and RELA during B‐cell generation and antigen‐dependent B‐cell activation have not been determined in vivo. To clarify this issue, we crossed mice with conditional rel and rela alleles individually or in combination to mice that express Cre‐recombinase in B cells. We here report that, whereas single deletion of rel or rela did not impair mature B‐cell generation and maintenance, their simultaneous deletion led to a dramatic reduction of follicular and marginal zone B cells. Upon T cell‐dependent immunization, B cell‐specific deletion of the c‐REL subunit alone abrogated the formation of germinal centers (GCs), whereas rela deletion did not affect GC formation. T‐independent responses were strongly impaired in mice with B cell‐specific deletion of rel, and only modestly in mice with RELA‐deficient B cells. Our findings identify differential requirements for the canonical NF‐κB subunits c‐REL and RELA at distinct stages of mature B‐cell development. The subunits are jointly required for the generation of mature B cells. During antigen‐dependent B‐cell activation, c‐REL is the critical subunit required for the initiation of the GC reaction and for optimal T‐independent antibody responses, with RELA being largely dispensable at this stage.