Inhibition of LTßR signalling activates WNT-induced regeneration in lung.

Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.

The helicase-like transcription factor redirects the autophagic flux and restricts human T cell leukemia virus type 1 infection.

Retroviruses and their host have coevolved in a delicate balance between viral replication and survival of the infected cell. In this equilibrium, restriction factors expressed by infected cells control different steps of retroviral replication such as entry, uncoating, nuclear import, expression, or budding. Here, we describe a mechanism of restriction against human T cell leukemia virus type 1 (HTLV-1) by the helicase-like transcription factor (HLTF). We show that RNA and protein levels of HLTF are reduced in primary T cells of HTLV-1-infected subjects, suggesting a clinical relevance. We further demonstrate that the viral oncogene Tax represses HLTF transcription via the Enhancer of zeste homolog 2 methyltransferase of the Polycomb repressive complex 2. The Tax protein also directly interacts with HLTF and induces its proteasomal degradation. RNA interference and gene transduction in HTLV-1-infected T cells derived from patients indicate that HLTF is a restriction factor. Restoring the normal levels of HLTF expression induces the dispersal of the Golgi apparatus and overproduction of secretory granules. By synergizing with Tax-mediated NF-κB activation, physiologically relevant levels of HLTF intensify the autophagic flux. Increased vesicular trafficking leads to an enlargement of the lysosomes and the production of large vacuoles containing viral particles. HLTF induction in HTLV-1-infected cells significantly increases the percentage of defective virions. In conclusion, HLTF-mediated activation of the autophagic flux blunts the infectious replication cycle of HTLV-1, revealing an original mode of viral restriction.

Novel Z-DNA binding domains in giant viruses.

Z-nucleic acid structures play vital roles in cellular processes and have implications in innate immunity due to their recognition by Zα domains containing proteins (Z-DNA/Z-RNA binding proteins, ZBPs). Although Zα domains have been identified in six proteins, including viral E3L, ORF112, and I73R, as well as, cellular ADAR1, ZBP1, and PKZ, their prevalence across living organisms remains largely unexplored. In this study, we introduce a computational approach to predict Zα domains, leading to the revelation of previously unidentified Zα domain-containing proteins in eukaryotic organisms, including non-metazoan species. Our findings encompass the discovery of new ZBPs in previously unexplored giant viruses, members of the Nucleocytoviricota phylum. Through experimental validation, we confirm the Zα functionality of select proteins, establishing their capability to induce the B-to-Z conversion. Additionally, we identify Zα-like domains within bacterial proteins. While these domains share certain features with Zα domains, they lack the ability to bind to Z-nucleic acids or facilitate the B-to-Z DNA conversion. Our findings significantly expand the ZBP family across a wide spectrum of organisms and raise intriguing questions about the evolutionary origins of Zα-containing proteins. Moreover, our study offers fresh perspectives on the functional significance of Zα domains in virus sensing and innate immunity and opens avenues for exploring hitherto undiscovered functions of ZBPs.

NF-κB-Independent Role of IKKα/IKKß in Preventing RIPK1 Kinase-Dependent Apoptotic and Necroptotic Cell Death during TNF Signaling.

TNF is a master pro-inflammatory cytokine. Activation of TNFR1 by TNF can result in both RIPK1-independent apoptosis and RIPK1 kinase-dependent apoptosis or necroptosis. These cell death outcomes are regulated by two distinct checkpoints during TNFR1 signaling. TNF-mediated NF-κB-dependent induction of pro-survival or anti-apoptotic molecules is a well-known late checkpoint in the pathway, protecting cells from RIPK1-independent death. On the other hand, the molecular mechanism regulating the contribution of RIPK1 to cell death is far less understood. We demonstrate here that the IKK complex phosphorylates RIPK1 at TNFR1 complex I and protects cells from RIPK1 kinase-dependent death, independent of its function in NF-κB activation. We provide in vitro and in vivo evidence that inhibition of IKKα/IKKß or its upstream activators sensitizes cells to death by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We therefore report on an unexpected, NF-κB-independent role for the IKK complex in protecting cells from RIPK1-dependent death downstream of TNFR1.

Hypoxia-Inducible Factor 1 Alpha-Mediated RelB/APOBEC3B Down-regulation Allows Hepatitis B Virus Persistence.

BACKGROUND AND AIMS: Therapeutic strategies against HBV focus, among others, on the activation of the immune system to enable the infected host to eliminate HBV. Hypoxia-inducible factor 1 alpha (HIF1α) stabilization has been associated with impaired immune responses. HBV pathogenesis triggers chronic hepatitis-related scaring, leading inter alia to modulation of liver oxygenation and transient immune activation, both factors playing a role in HIF1α stabilization. APPROACH AND RESULTS: We addressed whether HIF1α interferes with immune-mediated induction of the cytidine deaminase, apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B; A3B), and subsequent covalently closed circular DNA (cccDNA) decay. Liver biopsies of chronic HBV (CHB) patients were analyzed by immunohistochemistry and in situ hybridization. The effect of HIF1α induction/stabilization on differentiated HepaRG or mice ± HBV ± LTßR-agonist (BS1) was assessed in vitro and in vivo. Induction of A3B and subsequent effects were analyzed by RT-qPCR, immunoblotting, chromatin immunoprecipitation, immunocytochemistry, and mass spectrometry. Analyzing CHB highlighted that areas with high HIF1α levels and low A3B expression correlated with high HBcAg, potentially representing a reservoir for HBV survival in immune-active patients. In vitro, HIF1α stabilization strongly impaired A3B expression and anti-HBV effect. Interestingly, HIF1α knockdown was sufficient to rescue the inhibition of A3B up-regulation and -mediated antiviral effects, whereas HIF2α knockdown had no effect. HIF1α stabilization decreased the level of v-rel reticuloendotheliosis viral oncogene homolog B protein, but not its mRNA, which was confirmed in vivo. Noteworthy, this function of HIF1α was independent of its partner, aryl hydrocarbon receptor nuclear translocator. CONCLUSIONS: In conclusion, inhibiting HIF1α expression or stabilization represents an anti-HBV strategy in the context of immune-mediated A3B induction. High HIF1α, mediated by hypoxia or inflammation, offers a reservoir for HBV survival in vivo and should be considered as a restricting factor in the development of immune therapies.

A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control.

BACKGROUND & AIMS: Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver-resident macrophages. However, hepatocytes, the parenchymal cells of the liver, also possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct antiviral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-κB signaling (IkkßΔHep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/ß signaling-(IfnarΔHep), or interferon-α/ß signaling in myeloid cells-(IfnarΔMyel) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-κB signaling in hepatocytes. LCMV-triggered NF-κB activation in hepatocytes did not depend on Kupffer cells or TNFR1 signaling but rather on Toll-like receptor signaling. LCMV-infected IkkßΔHep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKß, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IkkßΔHep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IfnarΔHep mice, whereas IfnarΔMyel mice were able to control LCMV infection. Hepatocytic NF-κB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-α/ß-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-κB is a vital amplifier of interferon-α/ß signaling, which is pivotal for strong early ISG responses, immune cell infiltration and hepatic viral clearance. LAY SUMMARY: Innate immune cells have been ascribed a primary role in controlling viral clearance upon hepatic infections. We identified a novel dual role for NF-κB signaling in infected hepatocytes which was crucial for maximizing interferon responses and initiating adaptive immunity, thereby efficiently controlling hepatic virus replication.

RIPK4 activity in keratinocytes is controlled by the SCFß-TrCP ubiquitin ligase to maintain cortical actin organization.

RIPK4 is a key player in epidermal differentiation and barrier formation. RIPK4 signaling pathways controlling keratinocyte proliferation and differentiation depend on its kinase activity leading to Dvl2, Pkp1 and IRF6 phosphorylation and NF-κB activation. However, the mechanism regulating RIPK4 activity levels remains elusive. We show that cultured keratinocytes display constitutive active phosphorylated RIPK4 while PKC signaling can trigger RIPK4 activation in various non-keratinocyte cell lines, in which RIPK4 is present in a non-phosphorylated state. Interestingly, we identified the SCFß-TrCP ubiquitin E3 ligase complex responsible for regulating the active RIPK4 protein level. The SCFß-TrCP complex binds to a conserved phosphodegron motif in the intermediate domain of RIPK4, subsequently leading to K48-linked ubiquitinylation and degradation. The recruitment of ß-TrCP is dependent on RIPK4 activation and trans-autophosphorylation. ß-TrCP knock-down resulted in RIPK4-dependent formation of actin stress fibers, cell scattering and increased cell motility, suggesting that tight control of RIPK4 activity levels is crucial to maintain cell shape and behavior in keratinocytes.

Development of autoimmune pancreatitis is independent of CDKN1A/p21-mediated pancreatic inflammation.

OBJECTIVE: Chronic pancreatitis (CP) and autoimmune pancreatitis (AIP) are characterised by different inflammatory processes. If pancreatic inflammation is a prerequisite for autoimmunity is still unclear. AIP is considered mostly a T cell-mediated disease; however, in induction of CP, macrophages play a pivotal role. p21-a member of cyclin-dependent kinase inhibitors-can influence inflammatory processes, in particular can regulate T cell activation and promote macrophage development. We therefore examined the role of p21-mediated inflammation in AIP. DESIGN: We intercrossed lymphotoxin (LT) overexpressing mice (Tg(Ela1-LTa,b))-a model to study AIP development-with p21-deficient mice. Furthermore, we characterised p21 expression in human AIP and non-AIP specimens. RESULTS: p21 deficiency in LT mice (LTp21-/-) prevented early pancreatic injury and reduced inflammation. In acinar cells, diminished proliferation and abrogated activation of non-canonical nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathway was observed. In contrast, 12-month-old LT mice with and without p21 had similar inflammatory signatures and T-B cell infiltration. Interestingly, LT and LTp21-/- mice had comparable tertiary lymphoid organs (TLOs), autoantibodies and elevated IgG levels. However, acinar cell proliferation, acinar-to-ductal metaplasia and acinar non-canonical NF-κB pathway activation remained impaired in LTp21-/- pancreata. CONCLUSIONS: Our findings indicate that p21 is crucial for pancreatic inflammation in LT-driven pancreatic injury. p21 is involved in early acinar secretion of inflammatory mediators that attract innate immune cells. However, p21 is not essential for humoral immune response, accountable for autoimmunity. Remarkably, p21 renders acinar cells less susceptible to proliferation and transdifferentiation. We therefore suggest that AIP can also develop independent of chronic inflammatory processes.

Context-dependent roles for lymphotoxin-ß receptor signaling in cancer development.

The LTα1ß2 and LIGHT TNF superfamily cytokines exert pleiotropic physiological functions through the activation of their cognate lymphotoxin-ß receptor (LTßR). Interestingly, since the discovery of these proteins, accumulating evidence has pinpointed a role for LTßR signaling in carcinogenesis. Early studies have shown a potential anti-tumoral role in a subset of solid cancers either by triggering apoptosis in malignant cells or by eliciting an anti-tumor immune response. However, more recent studies provided robust evidence that LTßR signaling is also involved in diverse cell-intrinsic and microenvironment-dependent pro-oncogenic mechanisms, affecting several solid and hematological malignancies. Consequently, the usefulness of LTßR signaling axis blockade has been investigated as a potential therapeutic approach for cancer. Considering the seemingly opposite roles of LTßR signaling in diverse cancer types and their key implications for therapy, we here extensively review the different mechanisms by which LTßR activation affects carcinogenesis, focusing on the diverse contexts and different models assessed.

A role for APPL1 in TLR3/4-dependent TBK1 and IKKε activation in macrophages.

Endosomes have important roles in intracellular signal transduction as a sorting platform. Signaling cascades from TLR engagement to IRF3-dependent gene transcription rely on endosomes, yet the proteins that specifically recruit IRF3-activating molecules to them are poorly defined. We show that adaptor protein containing a pleckstrin-homology domain, a phosphotyrosine-binding domain, and a leucine zipper motif (APPL)1, an early endosomal protein, is required for both TRIF- and retinoic acid-inducible gene 1-dependent signaling cascades to induce IRF3 activation. APPL1, but not early endosome Ag 1, deficiency impairs IRF3 target gene expression upon engagement of both TLR3 and TLR4 pathways, as well as in H1N1-infected macrophages. The IRF3-phosphorylating kinases TBK1 and IKKε are recruited to APPL1 endosomes in LPS-stimulated macrophages. Interestingly, APPL1 undergoes proteasome-mediated degradation through ERK1/2 to turn off signaling. APPL1 degradation is blocked when signaling through the endosome is inhibited by chloroquine or dynasore. Therefore, APPL1 endosomes are critical for IRF3-dependent gene expression in response to some viral and bacterial infections in macrophages. Those signaling pathways involve the signal-induced degradation of APPL1 to prevent aberrant IRF3-dependent gene expression linked to immune diseases.

DUSP3 Genetic Deletion Confers M2-like Macrophage-Dependent Tolerance to Septic Shock.

DUSP3 is a small dual-specificity protein phosphatase with an unknown physiological function. We report that DUSP3 is strongly expressed in human and mouse monocytes and macrophages, and that its deficiency in mice promotes tolerance to LPS-induced endotoxin shock and to polymicrobial septic shock after cecal ligation and puncture. By using adoptive transfer experiments, we demonstrate that resistance to endotoxin is macrophage dependent and transferable, and that this protection is associated with a striking increase of M2-like macrophages in DUSP3(-/-) mice in both the LPS and cecal ligation and puncture models. We show that the altered response of DUSP3(-/-) mice to sepsis is reflected in decreased TNF production and impaired ERK1/2 activation. Our results demonstrate that DUSP3 plays a key and nonredundant role as a regulator of innate immune responses by mechanisms involving the control of ERK1/2 activation, TNF secretion, and macrophage polarization.

Lymphotoxin-ß receptor in microenvironmental cells promotes the development of T-cell acute lymphoblastic leukaemia with cortical/mature immunophenotype.

Lymphotoxin-mediated activation of the lymphotoxin-ß receptor (LTßR; LTBR) has been implicated in cancer, but its role in T-cell acute lymphoblastic leukaemia (T-ALL) has remained elusive. Here we show that the genes encoding lymphotoxin (LT)-α and LTß (LTA, LTB) are expressed in T-ALL patient samples, mostly of the TAL/LMO molecular subtype, and in the TEL-JAK2 transgenic mouse model of cortical/mature T-ALL (Lta, Ltb). In these mice, expression of Lta and Ltb is elevated in early stage T-ALL. Surface LTα1 ß2 protein is expressed in primary mouse T-ALL cells, but only in the absence of microenvironmental LTßR interaction. Indeed, surface LT expression is suppressed in leukaemic cells contacting Ltbr-expressing but not Ltbr-deficient stromal cells, both in vitro and in vivo, thus indicating that dynamic surface LT expression in leukaemic cells depends on interaction with its receptor. Supporting the notion that LT signalling plays a role in T-ALL, inactivation of Ltbr results in a significant delay in TEL-JAK2-induced leukaemia onset. Moreover, young asymptomatic TEL-JAK2;Ltbr(-/-) mice present markedly less leukaemic thymocytes than age-matched TEL-JAK2;Ltbr(+/+) mice and interference with LTßR function at this early stage delayed T-ALL development. We conclude that LT expression by T-ALL cells activates LTßR signalling in thymic stromal cells, thus promoting leukaemogenesis.

Compound heterozygous mutations in the kinase domain of IKKα lead to immunodeficiency and immune dysregulation.

IKKα, encoded by CHUK , is crucial in the non-canonical NF-κB pathway and part of the IKK complex activating the canonical pathway alongside IKKß. Absence of IKKα cause fetal encasement syndrome in human, fatal in utero, while an impaired IKKα-NIK interaction was reported in a single patient and cause combined immunodeficiency. Here, we describe compound heterozygous variants in the kinase domain of IKKα in a female patient with hypogammaglobulinemia, recurrent lung infections, and Hay-Wells syndrome-like features. We showed that both variants were loss-of-function. Non-canonical NF-κB activation was profoundly diminished in stromal and immune cells while the canonical pathway was partially impaired. Reintroducing wild-type CHUK restored non-canonical NF-κB activation. The patient had neutralizing autoantibodies against type I IFN, akin to non-canonical NF-κB pathway deficiencies. Thus, this is the first case of bi-allelic CHUK mutations disrupting IKKα kinase function, broadening non-canonical NF-κB defect understanding and suggesting IKKα's role in canonical NF-κB target gene expression in human.

NF-κB-inducing kinase (NIK) is activated in pancreatic ß-cells but does not contribute to the development of diabetes.

The transcription factor nuclear factor-κB (NF-κB) has a key role in the pathogenesis of diabetes and its complications. Although activation of the canonical NF-κB pathway in ß-cells is generally deleterious, little is known about the role of the non-canonical NF-κB signalling and its main regulator, the NF-κB-inducing kinase (NIK), on pancreatic ß-cell survival and function. Previous studies based on models of NIK overexpression in pancreatic islet cells showed that NIK induced either spontaneous ß-cell death due to islet inflammation or glucose intolerance during diet-induced obesity (DIO) in mice. Therefore, NIK has been proposed as a potential target for diabetes therapy. However, no clear studies showed whether inhibition of NIK improves diabetes development. Here we show that genetic silencing of NIK in pancreatic ß-cells neither modifies diabetes incidence nor inflammatory responses in a mouse model of immune-mediated diabetes. Moreover, NIK silencing in DIO mice did not influence body weight gain, nor glucose metabolism. In vitro studies corroborated the in vivo findings in terms of ß-cell survival, function, and downstream gene regulation. Taken together, our data suggest that NIK activation is dispensable for the development of diabetes.

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.

Varicella-zoster virus modulates NF-kappaB recruitment on selected cellular promoters.

Intercellular adhesion molecule 1 (ICAM-1) expression is down-regulated in the center of cutaneous varicella lesions despite the expression of proinflammatory cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-alpha). To study the molecular basis of this down-regulation, the ICAM-1 induction of TNF-alpha was analyzed in varicella-zoster virus (VZV)-infected melanoma cells (MeWo), leading to the following observations: (i) VZV inhibits the stimulation of icam-1 mRNA synthesis; (ii) despite VZV-induced nuclear translocation of p65, p52, and c-Rel, p50 does not translocate in response to TNF-alpha; (iii) the nuclear p65 present in VZV-infected cells is no longer associated with p50 and is unable to bind the proximal NF-kappaB site of the icam-1 promoter, despite an increased acetylation and accessibility of the promoter in response to TNF-alpha; and (iv) VZV induces the nuclear accumulation of the NF-kappaB inhibitor p100. VZV also inhibits icam-1 stimulation of TNF-alpha by strongly reducing NF-kappaB nuclear translocation in MRC5 fibroblasts. Taken together, these data show that VZV interferes with several aspects of the immune response by inhibiting NF-kappaB binding and the expression of target genes. Targeting NF-kappaB activation, which plays a central role in innate and adaptive immune responses, leads to obvious advantages for the virus, particularly in melanocytes, which are a site of viral replication in the skin.

The prevention of spontaneous apoptosis of follicular lymphoma B cells by a follicular dendritic cell line: involvement of caspase-3, caspase-8 and c-FLIP.

BACKGROUND: Follicular lymphoma, the neoplastic counterpart of germinal center B cells, typically recapitulates a follicular architecture. Several observations point to the crucial role of the cellular microenvironment in the development and/or progression of follicular lymphoma cells in vivo. The aim of our study was to characterize the spontaneous apoptosis of follicular lymphoma cells in vitro, and the modulation of this apoptosis by follicular dendritic cells. DESIGN AND METHODS: We used a cell line derived from follicular dendritic cells to model the functional interactions of these cells and lymphoma cells in co-culture. Follicular lymphoma cells were isolated from tissue biopsies. Apoptosis was quantified by flow cytometry and apoptotic pathways were investigated by western blotting. RESULTS: The spontaneous apoptosis of follicular lymphoma cells in vitro involves the activation of caspases-3 and -8 but not of caspase-9, occurs despite persistent high levels of BCL-2 and MCL-1, and is associated with down-regulation of c-FLIP(L). Spontaneous apoptosis of follicular lymphoma cells is partially prevented by co-culture with the follicular dendritic cells, which prevents activation of caspase-8, caspase-3 and induces an upregulation of c-FLIP(L). Using neutralizing antibodies, we demonstrated that interactions involving CD54 (ICAM-1), CD106 (VCAM-1) and CD40 are implicated in this biological process. CONCLUSIONS: Follicular dendritic cells constitute a useful tool to study the functional interactions between follicular lymphoma cells and follicular dendritic cells in vitro. Understanding the molecular mechanisms involved in these protective interactions may lead to the identification of therapeutic agents that might suppress the survival and growth of follicular lymphoma cells.

Further insights in the mechanisms of interleukin-1beta stimulation of osteoprotegerin in osteoblast-like cells.

UNLABELLED: The mechanisms of IL-1beta stimulation of OPG were studied in more detail. Whereas p38 and ERK activation was confirmed to be needed, NF-kappaB was not necessary for this regulation. We also found that OPG production after IL-1beta stimulation was not sufficient to block TRAIL-induced apoptosis in MG-63 cells. INTRODUCTION: Osteoprotegerin (OPG) plays a key role in the regulation of bone resorption and is stimulated by interleukin (IL)-1beta. Herein, we defined the mechanisms of IL-1beta stimulation of OPG focusing on the potential involvement of MAPK and NF-kappaB. We also examined whether OPG production in response to IL-1beta influences TRAIL-induced apoptosis in MG-63 cells. MATERIALS AND METHODS: OPG mRNA levels in MG-63 cells were quantified by real-time RT-PCR and protein levels of OPG and IL-6 by ELISA. Cell viability was assessed using the methyltetrazidium salt (MTS) reduction assay. The role of the MAPK pathway was studied by both Western blotting and the use of specific chemical inhibitors. NF-kappaB function was studied using BAY 11-7085 and by siRNA transfection to inhibit p65 synthesis. Transcription mechanisms were analyzed by transiently transfecting MG-63 cells with OPG promoter constructs. Post-transcriptional effects were examined by using cycloheximide and actinomycin D. RESULTS: MG-63 cells treatment with IL-1beta resulted in the phosphorylation of c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). The use of the specific inhibitors showed that p38 and ERK but not JNK were needed for IL-1beta-induced OPG production. In contrast, NF-kappaB was not essential for IL-1beta induction of OPG. We also showed a small transcriptional and a possible post-transcriptional or translational regulation of OPG by IL-1beta. Exogenous OPG blocked TRAIL-induced apoptosis, but IL-1beta induction of OPG did not influence TRAIL-induced cell death. CONCLUSIONS: IL-1beta stimulates OPG production by mechanisms dependent on p38 and ERK. In contrast, NF-kappaB was not essential for this regulation. Although the relevance of IL-1beta stimulation of OPG is still not fully understood, our data showed that IL-1beta stimulation of OPG does not modify TRAIL-induced cell death.

Egr family members regulate nonlymphoid expression of Fas ligand, TRAIL, and tumor necrosis factor during immune responses.

The Fas ligand (FasL)/Fas pathway is crucial for homeostasis of the immune system and peripheral tolerance. Peripheral lymphocyte deletion involves FasL/Fas in at least two ways: coexpression of both Fas and its ligand on T cells, leading to activation-induced cell death, and expression of FasL by nonlymphoid cells, such as intestinal epithelial cells (IEC), that kill Fas-positive T cells. We demonstrate here that superantigen Staphylococcus enterotoxin B (SEB) induced a dramatic upregulation of FasL, TRAIL, and TNF mRNA expression and function in IEC from BALB/c and C57BL/6 mice. Using adoptive transfer in which CD4(+) T cells from OT-2 T-cell receptor transgenic mice were transferred into recipients, we observed an induction in IEC of FasL, TRAIL, and TNF mRNA after administration of antigen. Specific Egr-binding sites have been identified in the 5' promoter region of the FasL gene, and Egr-1, Egr-2, and Egr-3 mRNA in IEC from mice treated with SEB and from transgenic OT-2 mice after administration of antigen was upregulated. Overexpression of Egr-2 and Egr-3 induced endogenous ligand upregulation that was inhibited by overexpression of Egr-specific inhibitor Nab1. These results support a role for Egr family members in nonlymphoid expression of FasL, TRAIL, and TNF.