Control of APOBEC3B induction and cccDNA decay by NF-κB and miR-138-5p.

BACKGROUND & AIMS: Immune-mediated induction of cytidine deaminase APOBEC3B (A3B) expression leads to HBV covalently closed circular DNA (cccDNA) decay. Here, we aimed to decipher the signalling pathway(s) and regulatory mechanism(s) involved in A3B induction and related HBV control. METHODS: Differentiated HepaRG cells (dHepaRG) knocked-down for NF-κB signalling components, transfected with siRNA or micro RNAs (miRNA), and primary human hepatocytes ± HBV or HBVΔX or HBV-RFP, were treated with lymphotoxin beta receptor (LTßR)-agonist (BS1). The biological outcomes were analysed by reverse transcriptase-qPCR, immunoblotting, luciferase activity, chromatin immune precipitation, electrophoretic mobility-shift assay, targeted-bisulfite-, miRNA-, RNA-, genome-sequencing, and mass-spectrometry. RESULTS: We found that canonical and non-canonical NF-κB signalling pathways are mandatory for A3B induction and anti-HBV effects. The degree of immune-mediated A3B production is independent of A3B promoter demethylation but is controlled post-transcriptionally by the miRNA 138-5p expression (hsa-miR-138-5p), promoting A3B mRNA decay. Hsa-miR-138-5p over-expression reduced A3B levels and its antiviral effects. Of note, established infection inhibited BS1-induced A3B expression through epigenetic modulation of A3B promoter. Twelve days of treatment with a LTßR-specific agonist BS1 is sufficient to reduce the cccDNA pool by 80% without inducing significant damages to a subset of cancer-related host genes. Interestingly, the A3B-mediated effect on HBV is independent of the transcriptional activity of cccDNA as well as on rcDNA synthesis. CONCLUSIONS: Altogether, A3B represents the only described enzyme to target both transcriptionally active and inactive cccDNA. Thus, inhibiting hsa-miR-138-5p expression should be considered in the combinatorial design of new therapies against HBV, especially in the context of immune-mediated A3B induction. LAY SUMMARY: Immune-mediated induction of cytidine deaminase APOBEC3B is transcriptionally regulated by NF-κB signalling and post-transcriptionally downregulated by hsa-miR-138-5p expression, leading to cccDNA decay. Timely controlled APOBEC3B-mediated cccDNA decay occurs independently of cccDNA transcriptional activity and without damage to a subset of cancer-related genes. Thus, APOBEC3B-mediated cccDNA decay could offer an efficient therapeutic alternative to target hepatitis B virus chronic infection.

NF-kappaB inducing kinase (NIK) inhibitors: identification of new scaffolds using virtual screening.

As a wide variety of pro-inflammatory cytokines are involved in the development of rheumatoid arthritis (RA), there is an urgent need for the discovery of novel therapeutic strategies. Among these, the inhibition of the NF-kappaB inducing kinase (NIK), a key enzyme of the NF-kappaB alternative pathway activation, represents a potential interesting approach. In fact, NIK is involved downstream of many tumor necrosis factor receptors (TNFR) like CD40, RANK or LTbetaR, implicated in the pathogenesis of RA. But, up to now, the number of reported putative NIK inhibitors is extremely limited. In this work, we report a virtual screening (VS) study combining various filters including high-throughput docking using a 3D-homology model and ranking by using different scoring functions. This work led to the identification of two molecular fragments, 4H-isoquinoline-1,3-dione (5) and 2,7-naphthydrine-1,3,6,8-tetrone (6) which inhibit NIK with an IC(50) value of 51 and 90 microM, respectively. This study opens new perspectives in the field of the NF-kappaB alternative pathway inhibition.

Role of IKK and ERK pathways in intrinsic inflammation of cystic fibrosis airways.

In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality and may precede bacterial colonization. The aim of the present study was to investigate the molecular mechanisms underlying intrinsic inflammation in cystic fibrosis airways. Using different cystic fibrosis cell models, we first demonstrated that, beside a high constitutive nuclear factor of kappaB (NF-kappaB) activity, CF cells showed a higher activator protein-1 (AP-1) activity as compared to their respective control cells. Gene expression profiles, confirmed by RT-PCR and ELISA, showed over-expression of numerous NF-kappaB and AP-1-dependent pro-inflammatory genes in CF cells in comparison with control cells. Activation of NF-kappaB was correlated with higher inhibitor of kappaB kinase (IKK) activity. In addition, Bio-plex phosphoprotein assays revealed higher extracellular signal-regulated kinase (ERK) phosphorylation in CFT-2 cells. Inhibition of this kinase strongly decreased expression of pro-inflammatory genes coding for growth-regulated proteins (Gro-alpha, Gro-beta and Gro-gamma) and interleukins (IL-1beta, IL-6 and IL-8). Moreover, inhibition of secreted interleukin-1beta (IL-1beta) and basic fibroblast growth factor (bFGF) with neutralizing antibodies reduced pro-inflammatory gene expression. Our data thus demonstrated for the first time that the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) at the plasma membrane leads to an intrinsic AP-1, in addition to NF-kappaB, activity and consequently to a pro-inflammatory state sustained through autocrine factors such as IL-1beta and bFGF.

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Methods to assess the activation of the alternative (noncanonical) NF-κB pathway by non-death TNF receptors.

The alternative or noncanonical NF-κB pathway regulates the generation of p52-containing NF-κB dimers (e.g., p52/RelB) through a partial degradation (called processing) of the precursor p100 into p52. This pathway is activated by a subset of non-death TNF receptor members, which ultimately activate two kinases: NIK (NF-κB-Inducing Kinase) and IKKα (Inhibitor of κB Kinase alpha). These kinases create a phosphodegron for the E3 ligase SCF-ß-TrCP that covalently binds K48-linked polyubiquitin chain onto p100 prior to its proteasomal processing. The resulting p52-containing complexes translocate into the nucleus to activate target genes involved in secondary lymphoid organ development, B cell survival or in osteoclastogenesis. We describe in this chapter straightforward methods to monitor the activation of the alternative NF-κB pathway. These methods uncover cytosolic and nuclear biochemical modifications of key proteins of the alternative NF-κB pathway required prior to the transcription of NF-κB target genes.

Noncanonical NF-κB signaling is limited by classical NF-κB activity.

Precise regulation of nuclear factor κB (NF-κB) signaling is crucial for normal immune responses, and defective NF-κB activity underlies a range of immunodeficiencies. NF-κB is activated through two signaling cascades: the classical and noncanonical pathways. The classical pathway requires inhibitor of κB kinase ß (IKKß) and NF-κB essential modulator (NEMO), and hypomorphic mutations in the gene encoding NEMO (ikbkg) lead to inherited immunodeficiencies, collectively termed NEMO-ID. Noncanonical NF-κB activation requires NF-κB-inducing kinase (NIK) and IKKα, but not NEMO. We found that noncanonical NF-κB was basally active in peripheral blood mononuclear cells from NEMO-ID patients and that noncanonical NF-κB signaling was similarly enhanced in cell lines lacking functional NEMO. NIK, which normally undergoes constitutive degradation, was aberrantly present in resting NEMO-deficient cells, and regulation of its abundance was rescued by reconstitution with full-length NEMO, but not a mutant NEMO protein unable to physically associate with IKKα or IKKß. Binding of NEMO to IKKα was not required for ligand-dependent stabilization of NIK or noncanonical NF-κB signaling. Rather, an intact and functional IKK complex was essential to suppress basal NIK activity in unstimulated cells. Despite interacting with IKKα and IKKß to form an IKK complex, NEMO mutants associated with immunodeficiency failed to rescue classical NF-κB signaling or reverse the accumulation of NIK. Together, these findings identify a crucial role for classical NF-κB activity in the suppression of basal noncanonical NF-κB signaling.

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.

Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-ß receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-ß receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases-for example, by lymphotoxin-ß receptor activation-allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.

Biology and signal transduction pathways of the Lymphotoxin-αß/LTßR system.

This review focuses on the biological functions and signalling pathways activated by Lymphotoxin α (LTα)/Lymphotoxin ß (LTß) and their receptor LTßR. Genetic mouse models shed light on crucial roles for LT/LTßR to build and to maintain the architecture of lymphoid organs and to ensure an adapted immune response against invading pathogens. However, chronic inflammation, autoimmunity, cell death or cancer development are disorders that occur when the LT/LTßR system is twisted. Biological inhibitors, such as antagonist antibodies or decoy receptors, have been developed and used in clinical trials for diseases associated to the LT/LTßR system. Recent progress in the understanding of cellular trafficking and NF-κB signalling pathways downstream of LTα/LTß may bring new opportunities to develop therapeutics that target the pathological functions of these cytokines.

Induction of the alternative NF-κB pathway by lymphotoxin αß (LTαß) relies on internalization of LTß receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin ß receptor (LTßR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTßR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTßR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTßR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTßR cellular trafficking as a process required for specific biological functions of NF-κB.

Pyrazolo[4,3-c]isoquinolines as potential inhibitors of NF-kappaB activation.

In this work, we aimed to build a 3D-model of NIK and to study the binding of pyrazolo[4,3-c]isoquinolines with a view to highlight the structural elements responsible for their inhibitory potency. However, in the course of this work, we unexpectedly found that the pyrazolo[4,3-c]isoquinolines initially reported as NIK inhibitors were neither inhibitors of this enzyme nor of the alternative NF-kappaB pathway, but were in fact inhibitors of another kinase, the TGF-beta activated kinase 1 (TAK1) which is involved in the classical NF-kappaB pathway.