Biochemical and functional characterization of TNF family ligands BAFF and APRIL

The TNF family ligands BAFF, APRIL and their heteromers regulate maturation and survival of B cells. BAFF and APRIL share the receptors TACI and BCMA. BAFF additionally binds to BAFFR, while APRIL binds to proteoglycans. Belimumab is an anti-BAFF antibody approved for the treatment of systemic lupus erythematosus (SLE), while the decoy receptor atacicept (TACI), developed to treat SLE and IgA nephropathy, targets both BAFF and APRIL. We combined genetic and pharmacological approaches to identify APRIL-BCMA, APRIL- TACI, and BAFF-TACI as three redundant pairs of ligand-receptor interactions involved in the maintenance of mouse plasma cells in the bone marrow and IgA-secreting cells in the gut. The existence of yet-to-be characterized IgG-producing cells, which do not require TACI and BCMA, and could be BAFF- and APRIL-independent, is also suggested. The most active form of BAFF is an ordered, capsid-like 60-mer structure. Unlike belimumab, atacicept can block the 60-mer. We found that adult human serum irreversibly dissociated BAFF 60-mer into 3-mer, explaining why BAFF 60-mer had never been identified in vivo. In cord blood, where BAFF levels were higher, and the dissociating activity was lower than in adult serum, a high molecular weight form of BAFF, with some attributes of BAFF 60-mer, was consistently detected. The nature of the 60-mer-dissociating activity of adult serum and the possible function of the high molecular weight form of BAFF in fetuses remain to be investigated. We next characterized that atacicept administered in healthy subjects or SLE patients bound and accumulated BAFF and BAFF/APRIL heteromers, but did not accumulate APRIL. The activity and protein levels of endogenous APRIL in the human circulation were further studied. We concluded that most of the APRIL present in serum is released by activated platelets after blood sampling, suggesting a possible implication of APRIL in coagulation. In addition, we found that the most frequently used commercial ELISA for human APRIL measures a form of APRIL unable to bind TACI and BCMA. Low levels of this non- conventional form of APRIL, “nc-APRIL”, correlated with a bad prognosis in patients diagnosed with atherosclerotic plaque in a 12-year follow-up study. Mechanistically, APRIL binds the negatively charged heparan sulfate proteoglycans (HSPGs) in arteries, which may limit retention of low-density lipoproteins (LDLs) in the subendothelial area and delay atherosclerosis progression. These studies extend our knowledge on the redundant role of BAFF and APRIL for plasma cell survival, and open new research areas for the role of two forms of APRIL in platelet biology and cardiovascular diseases. -- BAFF, APRIL et leurs hétéromères régulent la maturation et la survie des cellules B en liant les récepteurs TACI et BCMA. BAFF lie également BAFFR, tandis qu'APRIL se lie aux protéoglycans. Belimumab est un anticorps anti-BAFF approuvé pour le traitement du lupus érythémateux disséminé (LED), tandis qu’atacicept (TACI soluble), un récepteur soluble destiné à traiter le LED et la néphropathie à IgA, cible à la fois BAFF et APRIL. Nos approches génétiques et pharmacologiques identifient trois paires de ligand-récepteur fonctionnelles pour les plasmocytes de souris dans la moelle osseuse et l'intestin: APRIL- BCMA, APRIL-TACI et BAFF-TACI. L'existence de cellules productrices d'IgG qui ne dépendent ni de TACI ni de BCMA, et peut-être pas non plus de BAFF et d'APRIL, est également suggérée. La forme la plus active de BAFF est un 60-mer de structure bien définie. Atacicept bloque le 60-mer bien plus efficacement que belimumab. Nous avons constaté que le sérum humain adulte dissocie irréversiblement le BAFF 60-mer en 3-mer, expliquant pourquoi le BAFF 60- mer n'avait jamais été identifié in vivo. Dans le sang ombilical où les niveaux de BAFF sont plus élevés et l'activité dissociative plus faible que dans le sérum adulte, on trouve une forme de poids moléculaire élevé de BAFF dotée de plusieurs attributs du BAFF 60-mer. La dissociation des 60-mer chez l’adulte et la fonction du BAFF de haut poids moléculaire chez les fœtus restent à étudier. Nous avons ensuite déterminé que l'atacicept administré à l’être humain se sature de BAFF et d’hétéromères BAFF/APRIL, sans accumuler d’APRIL. L’étude de l’origine d’APRIL nous a alors appris que la majorité d’APRIL dans le sérum humain provient de l’activation des plaquettes sanguines activées après le prélèvement sanguin, suggérant une implication possible de l'APRIL dans la coagulation. En outre, nous avons constaté que l'ELISA commercial le plus fréquemment utilisé pour mesurer l'APRIL humain reconnaît une forme jusqu’alors inconnue et non conventionnelle d'APRIL "nc-APRIL", incapable de lier TACI et BCMA mais qui corrèle fortement avec un risque plus faible de décès chez les patients diagnostiqués avec une plaque athéromateuse dans une étude avec 12 ans du suivi. Le mécanisme pourrait être qu’en liant les protéoglycans dans les artères, APRIL limite la rétention sous-endothéliale des lipoprotéines de basse densité (LDL), retardant la progression de l'athérosclérose. Ces études étendent les connaissances sur le rôle de BAFF et d'APRIL pour la survie des plasmocytes, et ouvrent un nouveau domaine de recherche sur le rôle de deux formes d'APRIL dans la biologie des plaquettes et les maladies cardiovasculaires

A case–control study of bisphenol A and endometrioma among subgroup of Iranian women

Background: Endometriosis is a multifactorial hormonally related complex disease with unknown etiology. Epidemiologic data were suggested the possible effects of endocrine disrupting chemicals such as bisphenol A (BPA) on endometriosis. BPA is similar to endogenous estrogen and has the ability to interact with estrogen receptors and stimulate estrogen production. Our aim was to evaluate the relationship between urinary BPA concentrations in women with endometrioma. Materials and Methods: This case–control study consisted of fifty women who have been referred to gynecology and infertility center with endometrioma and were candidates for operative laparoscopy and ovarian cystectomy as cases. Fifty women who had not any evidence of endometrioma in clinical and ultrasound evaluation and came to the same clinic for routine check-up were selected as controls. One-time urine sample was collected after receiving informed consent before surgery and medical intervention. Total BPA in urine was measured with high-performance liquid chromatography method and detection limit was 0.33 ng/mL. Results: Percentage of urine samples containing BPA was 86% of cases and 82.4% of control. Urinary BPA showed a right-skewed distribution. The mean concentration of BPA was 5.53 ± 3.47 ng/mL and 1.43 ± 1.57 ng/mL in endometriosis and control group, respectively (P < 0.0001, Mann–Whitney U-test). The logistic regression showed that the odds ratio of the BPA was 1.74 (95% confidence interval: 1.40–2.16) after adjustment of age, parity, body mass index <30, and educational status. Conclusion: This study showed a positive association between urinary BPA concentrations and endometrioma. However, further large-scale studies are needed to confirm this hypothesis

The Association Between Bisphenol A and Polycystic Ovarian Syndrome: A Case-Control Study

Polycystic ovarian syndrome (PCOS) is an endocrine metabolic disorder with unclear etiopathogenesis among reproductive age women. Evidences show genetic susceptibility and environmental factors were associated with PCOS. The aim of this study was to find the association between urinary concentrations of Bisphenol-A as an endocrine disrupting chemical (EDC) and PCOS. A case-control study was conducted in 51 samples in each group. All cases were selected from women who diagnosed with PCOS at Gynecology and infertility center. The control group was selected from women who had clinical file in the center due to previous problem and came for routine check-up and pap smear. The participants were asked to collect a first-morning urine sample before any medical interventions. Total BPA in urine were measured with High Performance Liquid Chromatography (HPLC) method. Comparison of BPA level between two groups shows significantly higher level in PCOS group compared with control group (3.34 ± 2.63 vs 1.43 ± 1.57 ng/mL, P-value <0.001). Using logistic regression analysis, BPA as the main dependent variable, was significantly associated with PCOS with adjusted Odds Ratio (OR) equal to 1.53 (95% CI: 1.14-2.05, P-value =0.004). The results of this study indicated that BPA may play a major role in the PCOS pathogenesis. Further investigations with better design are necessary to confirm this association

An unappreciated cell survival-independent role for BAFF initiating chronic lymphocytic leukemia

BackgroundChronic Lymphocytic Leukemia (CLL) is characterized by the expansion of CD19+ CD5+ B cells but its origin remains debated. Mutated CLL may originate from post-germinal center B cells and unmutated CLL from CD5+ mature B cell precursors. Irrespective of precursor types, events initiating CLL remain unknown. The cytokines BAFF and APRIL each play a significant role in CLL cell survival and accumulation, but their involvement in disease initiation remains unclear.MethodsWe generated novel CLL models lacking BAFF or APRIL. In vivo experiments were conducted to explore the impact of BAFF or APRIL loss on leukemia initiation, progression, and dissemination. Additionally, RNA-seq and quantitative real-time PCR were performed to unveil the transcriptomic signature influenced by BAFF in CLL. The direct role of BAFF in controlling the expression of tumor-promoting genes was further assessed in patient-derived primary CLL cells ex-vivo.ResultsOur findings demonstrate a crucial role for BAFF, but not APRIL, in the initiation and dissemination of CLL cells. In the absence of BAFF or its receptor BAFF-R, the TCL1 transgene only increases CLL cell numbers in the peritoneal cavity, without dissemination into the periphery. While BAFF binding to BAFF-R is dispensable for peritoneal CLL cell survival, it is necessary to activate a tumor-promoting gene program, potentially linked to CLL initiation and progression. This direct role of BAFF in controlling the expression of tumor-promoting genes was confirmed in patient-derived primary CLL cells ex-vivo.ConclusionsOur study, involving both mouse and human CLL cells, suggests that BAFF might initiate CLL through mechanisms independent of cell survival. Combining current CLL therapies with BAFF inhibition could offer a dual benefit by reducing peripheral tumor burden and suppressing transformed CLL cell output

DataSheet_1_Kinetics of free and ligand-bound atacicept in human serum.pdf

BAFF (B cell activation factor of the TNF family/B lymphocyte stimulator, BLyS) and APRIL (a proliferation-inducing ligand) are targeted by atacicept, a decoy receptor consisting of the extracellular domain of TACI (transmembrane activator and calcium-modulator and cyclophilin (CAML) interactor) fused to the Fc portion of human IgG1. The purpose of the study was to characterize free and ligand-bound atacicept in humans. Total and active atacicept in serum of healthy volunteers receiving a single dose of subcutaneous atacicept or in patients treated weekly for one year were measured by ELISA, Western blot, or cell-based assays. Pharmacokinetics of free and bound atacicept were predicted based on total atacicept ELISA results. Persistence of complexes of purified atacicept bound to recombinant ligands was also monitored in mice. Results show that unbound or active atacicept in human serum exceeded 0.1 µg/ml for one week post administration, or throughout a 1-year treatment with weekly administrations. After a single administration of atacicept, endogenous BAFF bound to atacicept was detected after 8 h then increased about 100-fold within 2 to 4 weeks. Endogenous heteromers of BAFF and APRIL bound to atacicept also accumulated, but atacicept-APRIL complexes were not detected. In mice receiving intravenous injections of purified complexes pre-formed in vitro, atacicept-BAFF persisted longer (more than a week) than atacicept-APRIL (less than a day). Thus, only biologically inactive BAFF and BAFF-APRIL heteromers accumulate on atacicept in vivo. The measure of active atacicept provides further support for the once-weekly dosing regimen implemented in the clinical development of atacicept.</p

A loop region of BAFF controls B cell survival and regulates recognition by different inhibitors

The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors

APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.Hell