BAFF overexpression promotes anti-dsDNA B-cell maturation and antibody secretion

Overexpression of BAFF is believed to play an important role in systemic lupus erythematosus and elevated levels of serum BAFF have been found in lupus patients. Excess BAFF also leads to overproduction of anti-dsDNA antibodies and a lupus-like syndrome in mice. In the present study, we use mice transgenic for the R4A-Cμ (IgM) heavy chain of an anti-dsDNA antibody, to study the effects of BAFF overexpression on anti-dsDNA B-cell regulation. We observe that overexpression of BAFF promotes anti-dsDNA B-cell maturation and secretion of antibody and enriches for transgenic anti-dsDNA B cells in the marginal zone and follicular splenic compartments. In addition, our data suggests that BAFF rescues a subset of anti-dsDNA B cells from a regulatory checkpoint in the transitional stage of development.     

Cigarette smoke inhibits BAFF expression and mucosal immunoglobulin A responses in the lung during influenza virus infection

Background

It is incompletely understood how cigarette smoke (CS) exposure affects lung mucosal immune responses during viral respiratory infections. B cell activating factor belonging to the tumor necrosis factor family (BAFF) plays an important role in the induction of secretory immunoglobulin A (S-IgA) which is the main effector of the mucosal immune system. We therefore investigated the effects of CS exposure on BAFF expression and S-IgA responses in the lung during influenza virus infection.

Methods

Mice were exposed to CS and/or infected with influenza virus. Bronchoalveolar lavage fluid and lung compartments were analyzed for BAFF expression, influenza-specific S-IgA level and histological changes. Lung B cells were isolated and the activation-induced cytidine deaminase (Aicda) expression was determined. BEAS-2B cells were treated with CS extract (CSE), influenza virus, interferon beta or N-acetylcysteine and BAFF expression was measured.

Results

CS inhibited BAFF expression in the lung, particularly after long-term exposure. BAFF and S-IgA levels were increased during influenza virus infection. Three-month CS exposure prior to influenza virus infection resulted in reduced BAFF and S-IgA levels in the lung as well as augmented pulmonary inflammation on day 7 after infection. Prior CS exposure also caused decreased Aicda expression in lung B cells during infection. Neutralization of BAFF in the lung resulted in reduced S-IgA levels during influenza virus infection. CSE inhibited virus-mediated BAFF induction in a dose-dependent manner in BEAS-2B cells, while this inhibition of BAFF by CSE was prevented by pretreatment with the antioxidant N-acetylcysteine.

Conclusions

Our findings indicate that CS may hinder early mucosal IgA responses in the lung during influenza virus infection through oxidative inhibition of BAFF, which might contribute to the increased incidence and severity of viral infections in smokers.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0201-y) contains supplementary material, which is available to authorized users.     

The BAFF/APRIL system: Emerging functions beyond B cell biology and autoimmunity

The BAFF system plays a key role in the development of autoimmunity, especially in systemic lupus erythematosus (SLE). This often leads to the assumption that BAFF is mostly a B cell factor with a specific role in autoimmunity. Focus on BAFF and autoimmunity, driven by pharmaceutical successes with the recent approval of a novel targeted therapy Belimumab, has relegated other potential roles of BAFF to the background. Far from being SLE-specific, the BAFF system has a much broader relevance in infection, cancer and allergy. In this review, we provide the latest views on additional roles of the BAFF system in health and diseases, as well as an update on BAFF and autoimmunity, with particular focus on current clinical trials.     

Expression,characterization of recombinant human soluble BAFF secreted from CHO cell

B cell-activating factor of the TNF family (BAFF) is critical for B cell maturation and survival. Here, we constructed a stable CHO cell line, in which the expression level of soluble form of BAFF (sBAFF) was raised from 0.13 μg/ml to 0.55 μg/ml. Purified recombinant sBAFF from these CHO cells not only bound to its receptors but also co-stimulated the proliferation of human peripheral blood B lymphocyte in vitro. These results provided us with a useful basis for further studies about sBAFF-related research.     

BAFF inhibition: a new class of drugs for the treatment of autoimmunity

BAFF (BLyS) and APRIL are TNF-like cytokines that support survival and differentiation of B cells. Recent studies have discovered a role for BAFF in augmenting both innate and adaptive immune responses as well as in collaborating with other inflammatory cytokines to promote the activation and differentiation of effector immune cells. BAFF is an important pathogenic factor in lupus mouse models and BAFF inhibition successfully delays disease onset in these mice, although the responsiveness to BAFF inhibition varies among different strains. These results have led to the development of inhibitors targeting BAFF and APRIL in humans. An anti-BAFF antibody has shown significant but modest efficacy in two Phase III clinical trials for moderately active SLE and other inhibitors are being developed or at early stages of clinical testing.     

B‐cell‐activating factor code and human cytomegalovirus infection in renal transplant recipients

The objective of the present study was to explore the correlation between the BAFF signal and HCMV‐TLR activation in RTx recipients complicated by HCMV. Peripheral blood (anticoagulated by EDTA‐Na₂) and urine of 113 RTx recipients were collected; healthy volunteers were controlled. Urine HCMV‐DNA was detected by real‐time PCR. Recipients were classified into a positive group (>10,000 copies/mL urine) and a negative group (<10,000 copies/mL urine). ELISA results showed that sBAFF, sera anti‐HCMV pp65 immunoglobulin (Ig)G antibody, and total IgG all significantly increased in recipients with positive HCMV‐DNA (>10,000 copies/mL urine) (P < 0.05) compared with negative recipients (<10,000 copies/mL urine). In the positive group, HCMV‐DNA copies and total IgG positively correlated with sBAFF (r = 0.988 and 0.625, respectively) (P < 0.05). Luminex assay results suggested that the incidence of anti‐HLA I and II and MICA antibody obviously increased in positive recipients. The expression level of BAFF and BAFF‐R increased in positive recipients. A total of 88 particular genes—involved in TLR signaling pathways, NF‐κB signaling pathways, and cytokine‐cytokine receptor signaling pathways—were detected in real‐time PCR chip assay. A total of 46 genes were differentially expressed greater than two‐fold, and the expression characteristic of BAFF‐R was concordant with FACS results. Our findings are that activation of HCMV would induce or enhance the activation of BAFF code in RTx recipients, which may independently or cooperatively participate in renal allograft injury and decrease the long‐term outcome of renal allografts.     

High triiodothyronine levels induce myocardial hypertrophy via BAFF overexpression

Activated B cells contribute to heart diseases, and inhibition of B‐cell activating factor (BAFF) expression is an effective therapeutic target for heart diseases. Whether activated B cells participate in the development and progression of hyperthyroid heart disease, and what induces B cells activation in hyperthyroidism are unknown. The present study aimed to determine the roles of BAFF overexpression induced by high concentrations of triiodothyronine (T3) in the pathogenesis of hyperthyroid heart disease. Female C57BL/6J mice were subcutaneously injected with T3 for 6 weeks, and BAFF expression was inhibited using shRNA. Protein and mRNA expression of BAFF in mouse heart tissues evaluated via immunohistochemistry, western blotting and polymerase chain reaction (PCR). Proportions of B cells in mouse cardiac tissue lymphocytes were quantified via flow cytometry. Morphology and left ventricle function were assessed using pathological sections and echocardiography, respectively. Here, we demonstrate that compared with the control group, the proportion of myocardial B cells was larger in the T3 group; immunohistochemistry, western blotting and PCR analyses revealed increased protein and mRNA expression levels of TNF‐α and BAFF in heart tissues of the T3 group. Compared with the normal controls group, in the T3 group, the diameter of myocardial cells and some echocardiographic values significantly increased and hypertrophy and structural disorder were noticeable. Our results revealed that elevated levels of circulating T3 can promote the expression of BAFF in myocardial cells and can lead to B‐cell activation, an elevated inflammatory response and ventricular remodelling.     

Clinical associations of serum interleukin-17 in systemic lupus erythematosus

Introduction

Serum interleukin (IL)-17 concentrations have been reported to be increased in systemic lupus erythematosus (SLE), but associations with clinical characteristics are not well understood. We characterized clinical associations of serum IL-17 in SLE.

Methods

We quantified IL-17 in serum samples from 98 SLE patients studied cross-sectionally, and in 246 samples from 75 of these patients followed longitudinally over two years. Disease activity was recorded using the SLE Disease Activity Index (SLEDAI)-2k. Serum IL-6, migration inhibitory factor (MIF), and B cell activating factor of the tumour necrosis factor family (BAFF) were also measured in these samples.

Results

Serum IL-17 levels were significantly higher in SLE patients compared to healthy donors (P <0.0001). No correlation was observed between serum IL-17 and SLEDAI-2k, at baseline or during longitudinal follow-up. However, we observed that SLEDAI-2k was positively correlated with IL-17/IL-6 ratio. Serum IL-17 was significantly increased in SLE patients with central nervous system (CNS) disease (P = 0.0298). A strong correlation was observed between serum IL-17 and IL-6 (r = 0.62, P <0.0001), and this relationship was observed regardless of disease activity and persisted when integrating cytokine levels over the period observed (r = 0.66, P <0.0001). A strong correlation of serum IL-17 was also observed with serum BAFF (r = 0.64, P <0.0001), and MIF (r = 0.36, P = 0.0016).

Conclusions

Serum IL-17 concentration correlates poorly with SLE disease activity but is significantly elevated in patients with CNS disease. IL-17/IL-6 ratio may be more useful than IL-17 or IL-6 alone to characterize Th17-driven disease, such as SLE. The association of other cytokines with serum IL-17 suggests that IL-17 may drive activation of diverse immune pathways in SLE.     

First BAFF gene cloned from an aquatic mammal

The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species. Research into the immune system of the finless porpoise is essential to the protection of this species, but, to date, no genes coding for proteins from the tumor necrosis factor family (TNF family) have yet been reported from finless porpoises. The TNF B cell activating factor (BAFF) is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. It acts through its three receptors, BAFF-R, BCMA, and TACI. In the present study, the full-length cDNA of BAFF (designated NpBAFF) from the finless porpoise was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques, and its biological activities have been characterized. To our knowledge, this is the first report of any BAFF gene being cloned from an aquatic mammal. The full-length cDNA of NpBAFF consists of 1502 bases including an 852 bp open reading frame encoding 283 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known BAFF homologues. Sequence comparison indicated that the amino acid sequence of NpBAFF was very similar to its bovine (87.68%), porcine (76.33%), hircine (87.68%) and canine (82.19%) counterparts. The predicted three-dimensional (3D) structure of the NpsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its human counterpart. Phylogenetic analysis indicated that NpBAFF showed a notable homology with Artiodactyla BAFFs. The SUMO-NpsBAFF was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that NpsBAFF could bind to its receptors on B cells. In vitro, MTT assays indicated that SUMO-NpsBAFF could promote the survival or proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that NpBAFF plays an important role in the survival or proliferation of B cells and has functional cross-reactivity among cetaceans and other mammals. The present findings may provide valuable information for research into the immune system of the finless porpoise.     

ROS/Epac1-mediated Rap1/NF-kappaB activation is required for the expression of BAFF in Raw264.7 murine macrophages

B-cell activating factor (BAFF) plays a role for the maturation and the maintenance of B cells. Lipopolysaccharide (LPS) activates toll-like receptor 4 (TLR4)-dependent signal transduction, which resulted in BAFF expression through nuclear factor kappa B (NF-κB) activation. Here, we investigated whether BAFF expression could be regulated by p65 phosphorylation through the production of reactive oxygen species (ROS) or cyclic AMP (cAMP) in Raw264.7 murine macrophages. mBAFF expression was reduced by ROS scavengers and it was increased by dibutyl-cAMP, a cAMP analogue. mBAFF expression and mBAFF promoter activity were increased by co-transfection of p65 but they were reduced by p65-small interference (si) RNA. Serine (Ser) 276 phosphorylation of p65 was increased by LPS-mediated PKA activation or by the treatment with forskolin, adenylate cyclase activator and dibutyl-cAMP. In contrast, p65 phosphorylation at Ser276 was decreased by ROS scavengers. H₂O₂ increased intracellular cAMP concentration, significantly. While no increase in p65 phosphorylation at Ser276 was detected by the treatment with H₂O₂, CREB and p65 phosphorylation at Ser133 and Ser536 was observed, respectively. It implicates that p65 phosphorylation at Ser276 is independent of ROS-induced cAMP production. As another cAMP effector protein was cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor, NF-κB was activated by the treatment with 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT) that is an activator to Epac. Epac1-mediated Rap1 was activated by the treatment with H₂O₂ but it was inhibited by ROS scavengers. CPT induced p65 phosphorylation at both Ser276 and Ser536. CPT also increased not only mBAFF expression but mBAFF promoter activity. Data demonstrate that TLR4-mediated mBAFF expression was resulted from the crosstalk of p65 phosphorylation at Ser536 and Ser276 through ROS- and/or cAMP-mediated signal transduction. It suggests for the first time that ROS/Epac1-mediated Rap1/NF-κB pathway could be required for BAFF expression.     

Multiple Novel Classes of APRIL-specific Receptor-blocking Peptides Isolated by Phage Display

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC₅₀ values of 0.49-27 μM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL-receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.     

NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.     

Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies

The somatic mutations accumulated in variable and framework regions of antibodies produce structural changes that increase the affinity towards the antigen. This implies conformational and non covalent bonding changes at the paratope, as well as possible quaternary structure changes and rearrangements at the V_H-V_L interface. The consequences of the affinity maturation on the stability of the Fv domain were studied in a system composed of two closely related antibodies, F10.6.6 and D44.1, which recognize the same hen egg-white lysozyme (HEL) epitope. The mAb F10.6.6 has an affinity constant 700 times higher than D44.1, due to a higher surface complementarity to HEL. The structure of the free form of the Fab F10.6.6 presented here allows a comparative study of the conformational changes produced upon binding to antigen. By means of structural comparison, kinetics and thermodynamics of binding and stability studies on Fab and Fv fragments of both antibodies, we have determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the V_H-V_L interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen.     

cDNA cloning, recombinant expression and bioactivity of Père David's deer BAFF

B cell activating factor (BAFF), a member of the TNF family, is a critical cytokine for the survival, proliferation, maturation, and differentiation of B cells. In the present study, Père David's deer BAFF (miBAFF) was amplified from Elaphurus davidianus using RT-PCR. This is the first BAFF cloned from a member of Cervidae family. The open reading frame (ORF) of the miBAFF cDNA consists of 843 bases that encode a 280-amino acid protein bearing typical TNF homology domain. Sequence alignment shows that miBAFF shares 39.3%-97% sequence homology with the BAFF sequences of other mammals. Comparative protein modeling predicted that the 3D structure of the soluble mature portion of miBAFF (misBAFF) is very similar to that of human BAFF (hsBAFF). Recombinant misBAFF fused to a SUMO-tag was efficiently expressed in Escherichia coli BL21 (DE3) cells. The protein molecular weight of ~36 KDa was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified misBAFF was shown to promote the survival and proliferation of Père David's deer peripheral blood lymphocytes and mouse B cells. These results indicate that miBAFF plays an important role in the survival/proliferation of mouse B cells and, shows highly conserved evolutionarily, leading to functional cross-reactivity that exists between mouse and Père David's deer BAFF.     

Toll-like receptor 9 signaling has anti-inflammatory effects on the early phase of Helicobacter pylori-induced gastritis

Helicobacter pylori (H. pylori)-induced immune responses in the gastric mucosa are skewed toward T helper (Th) 1 phenotype, which is characterized by predominant production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by helper T cells. Toll-like receptors (TLRs) play an essential role in mucosal defense against microbes through the recognition of bacterial molecules. Among the members of the TLR family, TLR9 recognizes bacterial unmethylated CpG DNA sites, and signal transduction of TLR9 induces production of a variety of cytokines, including type-I IFN (IFN-α/β). We investigated the expression and role of TLR9 in H. pylori-induced gastritis in mice. Expression of TLR9 mRNA in the gastric tissue increased after infection with H. pylori. TLR9 was mainly expressed in the macrophages, dendritic cells, and CD3⁺ cells in the gastric mucosa. Neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA were higher in TLR9 knockout (KO) mice than in wild-type mice at 2 and 4 months after H. pylori inoculation. These differences in inflammatory parameters between H. pylori-infected wild-type and TLR9 KO mice disappeared 6 months after H. pylori inoculation. Expression of interleukin-4 mRNA, typical Th2 cytokine, in the gastric tissue did not differ between H. pylori-infected wild-type and TLR9 KO mice. Expression level of IFN-α/β mRNA in the TLR9 KO mice was lower than that in wild-type mice by 4 months after inoculation. Administration of IFN-α reduced H. pylori infection-induced increase in neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA in TLR9 KO mice. Our findings suggest that TLR9 signaling plays important roles in the suppression of H. pylori-induced gastritis in the early phase via downregulation of Th1-type cytokines modulated by IFN-α.     

Toll-like receptor 4-mediated cAMP production up-regulates B-cell activating factor expression in Raw264.7 macrophages

B-cell activating factor (BAFF) plays a role in the generation and the maintenance of mature B cells. Lipopolysaccharide (LPS) increased BAFF expression through the activation of toll-like receptor 4 (TLR4)-dependent signal transduction. Here, we investigated the mechanism of action on mouse BAFF (mBAFF) expression by cAMP production in Raw264.7 mouse macrophages. mBAFF expression was increased by the treatment with a cAMP analogue, dibutyryl-cAMP which is the activator of protein kinase A (PKA), cAMP effector protein. PKA activation was measured by the phosphorylation of cAMP-response element binding protein (CREB) on serine 133 (S133). cAMP production and CREB (S133) phosphorylation were augmented by LPS-stimulation. While mBAFF promoter activity was enhanced by the co-transfection with pS6-RSV-CREB, it was reduced by siRNA-CREB. PKA inhibitor, H-89, reduced CREB (S133) phosphorylation and mBAFF expression in control and LPS-stimulated macrophages. Another principal cAMP effector protein is cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. Epac was activated by the treatment with 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT), Epac activator, as judged by the measurement of Rap1 activation. Basal level of mBAFF expression was increased by CPT treatment. LPS-stimulated mBAFF expression was also slightly enhanced by co-treatment with CPT. In addition, dibutyryl-cAMP and CPT enhanced mBAFF expression in bone marrow-derived macrophages (BMDM). With these data, it suggests that the activation of PKA and cAMP/Epac1/Rap1 pathways could be required for basal mBAFF expression, as well as being up-regulated in the TLR4-induced mBAFF expression.     

High metal concentrations are required for self-association of synaptotagmin II

Several members of the synaptotagmin (syt) family of vesicle proteins have been proposed to act as Ca2+ sensors on synaptic vesicles. The mechanism by which calcium activates this class of proteins has been the subject of controversy, yet relatively few detailed biophysical studies have been reported on how isoforms other than syt I respond to divalent metal ions. Here, we report a series of studies on the response of syt II to a wide range of metal ions. Analytical ultracentrifugation studies demonstrate that Ca2+ induces protein dimerization upon exposure to 5 mM Ca2+. Whereas Ba2+, Mg2+, or Sr2+ do not potentiate self-association as strongly as Ca2+, Pb2+ triggers self-association of syt II at concentrations as low as 10 microM. Partial proteolysis studies suggest that the various divalent metals cause different changes in the conformation of the protein. The high calcium concentrations required for self-association of syt II suggest that the oligomerized state of this protein is not a critical intermediate in vesicle fusion; however, low-affinity calcium sites on syt II may play a critical role in buffering calcium at the presynaptic active zone. In addition, the high propensity of lead to oligomerize syt II offers a possible molecular explanation for how lead interferes with calcium-evoked neurotransmitter release.