Although acetylcholine is widely utilized in vertebrate nervous systems, nicotinic acetylcholine receptors (nAChRs), including the α9α10 subtype, also are expressed in a wide variety of non-neuronal cells. These cell types include cochlear hair cells, adrenal chromaffin cells and immune cells. α9α10 nAChRs present in these cells may respectively play roles in protection from noise-induced hearing loss, response to stress and neuroprotection. Despite these critical functions, there are few available selective ligands to confirm mechanistic hypothesis regarding the role of α9α10 nAChRs. Conus, has been a rich source of ligands for receptors and ion channels. Here, we identified Conus geographus venom as a lead source for a novel α9α10 antagonist. The active component was isolated and the encoding gene cloned. The peptide signal sequence and cysteine arrangement had the signature of the σ-conotoxin superfamily. Previously isolated σ-conotoxin GVIIIA, also from Conus geographus, targets the 5-HT3 receptor. In contrast, αS-GVIIIB blocked the α9α10 nAChR with an IC50 of 9.8 nM, yet was inactive at the 5-HT3 receptor. Pharmacological characterization of αS-GVIIIB shows that it is over 100-fold selective for the α9α10 nAChR compared to other nAChR subtypes. Thus, the S-superfamily represents a novel conotoxin scaffold for flexibly targeting a variety of receptor subtypes. Functional competition studies utilized distinct off-rate kinetics of conotoxins to identify the α10/α9 nAChR interface as the site of αS-GVIIIB binding; this adds to the importance of the (+) face of the α10 rather than the (+) face of the α9 nAChR subunit as critical to binding of α9α10-targeted conotoxins.
protein purification; oocyte; nicotinic receptor; alpha9 alpha10; conotoxin
The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the α-subunit of nicotinic acetylcholine receptors and in particular the homomeric α7 nicotinic receptor. We report the isolation and characterization of an α-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the α7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the α3β2 nAChR indicating that α-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of α3β2 nAChRs. α-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first α-conotoxin with higher affinity for the closely related receptor subtypes, α3β2 versus α6β2, and selectively blocks these two subtypes when compared with α2β2, α4β2, and α1β1δε nAChRs.
The α3β4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an α-4/6-conotoxin (CTx) TxID from Conus textile. The new toxin consists of 15 amino acid residues with two disulfide bonds. TxID was synthesized using solid phase methods and the synthetic peptide was functionally tested on nAChRs heterologously expressed in Xenopus laevis oocytes. TxID blocked rat α3β4 nAChRs with a 12.5 nM IC50, which places it amongst the most potent α3β4 nAChR antagonists. TxID also blocked the closely related α6/α3β4 with a 94 nM IC50 but showed little activity on other nAChR subtypes. NMR analysis showed that two major structural isomers exist in solution, one of which adopts a regular α-CTx fold but with different surface charge distribution to other 4/6 family members. α-CTx TxID is a novel tool with which to probe the structure and function of α3β4 nAChRs.
α-CTx; α3β 4 nAChRs; potent antagonist; NMR
On the lupus-prone MRL-lpr/lpr (MRL-lpr) background AM14 rheumatoid factor (RF) B cells are activated, differentiate into plasmablasts, and undergo somatic hypermutation outside of follicles. Using multiple strategies to impair T cells, we found that such AM14 B cell activation did not require T cells, but could be modulated by them. In vitro, the signaling adaptor MyD88 is required for IgG anti-chromatin to stimulate AM14 B cell proliferation when T cells are absent. However the roles of Toll-like receptors (TLRs) in AM14 B cell activation in vivo have not been investigated. We found that activation, expansion and differentiation of AM14 B cells depended on MyD88; however, mice lacking either TLR7 or TLR9 displayed partial defects, indicating complex roles for these receptors. T-independent activation of certain autoreactive B cells, which instead can gain stimuli via endogenous TLR ligands, may be the initial step in the generation of canonical autoantibodies.
Systemic lupus erythematosus is characterized by the production of autoantibodies against nucleic acid-associated Ags. We previously found that Tlr7 was required for anti-Sm and Tlr9 for anti-chromatin autoantibodies. Yet, although Tlr7 deficiency ameliorated disease, Tlr9 deficiency exacerbated it. Despite the mechanistic and clinical implications of this finding, it has yet to be elucidated. In this study, we characterize MRL/lpr lupus-prone mice genetically deficient in Tlr7, Tlr9, both Tlr7 and Tlr9, or Myd88 to test whether Tlr7 and Tlr9 function independently or instead regulate each other. We find that disease that is regulated by Tlr9 (and hence is worse in its absence) depends on Tlr7 for its manifestation. In addition, although Tlr7 and Tlr9 act in parallel pathways on different subsets of autoantibodies, Tlr9 also suppresses the production of Tlr7-dependent RNA-associated autoantibodies, suggesting previously unrecognized cross-regulation of autoantibody production as well. By comparing disease in mice deficient for Tlr7 and/or Tlr9 to those lacking Myd88, we also identify aspects of disease that have Tlr- and Myd88-independent components. These results suggest new models for how Tlr9 regulates and Tlr7 enhances disease and provide insight into aspects of autoimmune disease that are, and are not, influenced by TLR signals.
Nucleic acid reactive B cells frequently arise in the bone marrow but are tolerized by mechanisms including receptor editing, functional anergy, and/or deletion. TLR9, a sensor of endosomal dsDNA, both promotes and regulates systemic autoimmunity in vivo, but the precise nature of its apparently contradictory roles in autoimmunity remained unclear. Here, using the 3H9 anti-DNA BCR transgene in the autoimmune-prone MRL.Faslpr mouse model of systemic lupus erythematosus, we identify the stages at which TLR9 contributes to establishing and breaking B cell tolerance. Although TLR9 is dispensable for light chain editing during B cell development in the bone marrow, TLR9 limits anti-DNA B cell lifespan in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer lifespans and accumulate in the follicle, neither activated nor deleted. These cells retain some characteristics of anergic cells, in that they have elevated basal BCR signaling but impaired induced responses and downregulate their cell surface BCR expression. In contrast, while TLR9-intact anergic B cells accumulate near the T/B border, TLR9-deficient anti-DNA B cells are somewhat more dispersed throughout the follicle. Nonetheless, in older autoimmune-prone animals, TLR9 expression specifically within the B cell compartment is required for spontaneous peripheral activation of anti-DNA B cells and their differentiation into AFCs via an extrafollicular pathway. Thus, TLR9 has paradoxical roles in regulating anti-DNA B cells: it helps purge the peripheral repertoire of autoreactive cells yet is also required for their activation.
The AM14 rheumatoid factor (RF) transgenic (Tg) mouse has been valuable for studying how self-reactive B cells are regulated beyond central tolerance, because they remain ignorant in normal mice. AM14 B cell activation can be studied on autoimmune-prone strains or by inducing activation with IgG2a anti-chromatin antibodies. Despite the utility of conventional Ig-Tg mice, site-directed Ig Tg (sd-Tg) mice provide a more physiological model for B cell responses, allowing class switch and somatic hypermutation. We report here the creation of an AM14 sd-Tg mouse and describe its phenotype on both normal and autoimmune-prone backgrounds. AM14 sd-Tg B cells develop normally but remain unactivated on the BALB/c background, even after significant aging. In contrast, on the autoimmune prone strain MRL/lpr, AM14 sd-Tg B cells become activated and secrete large amounts of IgG RF antibody into the serum. Class-switched antibody forming cells were found in the spleen and bone marrow. IgG RF plasmablasts were also observed in extrafollicular clusters in the spleens of aged AM14 sd-Tg MRL/lpr mice. Class switch and antibody secretion were observed additionally in AM14 sd-Tg BALB/c B cells activated in vivo using IgG2a anti-chromatin antibodies. Development of IgG autoantibodies is a hallmark of severe autoimmunity, and is related to pathogenesis. Using the AM14 sd-Tg, we now show that switched autoantibody-forming cells develop robustly outside germinal centers, further confirming the extrafollicular expression of AID. This model will allow more physiological studies of B cell biology in the future, including memory responses marked by class switch.
plasmablast; isotype switch; lupus; extrafollicular response
Conotoxins (CTxs) selectively target a range of ion channels and receptors, making them widely used tools for probing nervous system function. Conotoxins have been previously grouped into superfamilies according to signal sequence and into families based on their cysteine framework and biological target. Here we describe the cloning and characterization of a new conotoxin, from Conus vexillum, named αB-conotoxin VxXXIVA. The peptide does not belong to any previously described conotoxin superfamily and its arrangement of Cys residues is unique among conopeptides. Moreover, in contrast to previously characterized conopeptide toxins, which are expressed initially as prepropeptide precursors with a signal sequence, a ‘‘pro’’ region, and the toxin-encoding region, the precursor sequence of αB-VxXXIVA lacks a ‘‘pro’’ region. The predicted 40-residue mature peptide, which contains four Cys, was synthesized in each of the three possible disulfide arrangements. Investigation of the mechanism of action of αB-VxXXIVA revealed that the peptide is a nicotinic acetylcholine receptor (nAChR) antagonist with greatest potency against the α9α10 subtype. 1H nuclear magnetic resonance (NMR) spectra indicated that all three αB-VxXXIVA isomers were poorly structured in aqueous solution. This was consistent with circular dichroism (CD) results which showed that the peptides were unstructured in buffer, but adopted partially helical conformations in aqueous trifluoroethanol (TFE) solution. The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics, and αB-VxXXIVA represents a novel ligand with which to probe the structure and function of this protein.
Conus species are characterized by their hyperdiverse toxins, encoded by a few gene superfamilies. Our phylogenies of the genus, based on mitochondrial genes, confirm previous results that C. californicus is highly divergent from all other species. Genetic and biochemical analysis of their venom peptides comprise the fifteen most abundant conopeptides and over 50 mature cDNA transcripts from the venom duct. Although C. californicus venom retains many of the general properties of other Conus species, they share only half of the toxin gene superfamilies found in other Conus species. Thus, in these two lineages, approximately half of the rapidly diversifying gene superfamilies originated after an early Tertiary split. Such results demonstrate that, unlike endogenously acting gene families, these genes are likely to be significantly more restricted in their phylogenetic distribution. In concordance with the evolutionary duistance of C. californicus from other species, there are aspects of prey-capture behavior and prey preferences of this species that diverges significantly from all other Conus.
biodiversity; accelerated evolution; gene superfamilies; cone snail; exogenomics; phylogenetic relationships
Synthetic oligonucleotides containing CpG motifs (CpG ODNs) have been shown to induce proliferation, differentiation and cytokine production in B cells, macrophages and DCs through a TLR9-dependent mechanism. A class (CpG-A) and B class (CpG-B) ODNs display distinct physical properties. CpG-A, but not CpG-B, can multimerize to form exceedingly large lattices. CpG-A cannot effectively activate B cells but does induce pDCs to produce high levels of IFNα, while CpG-B is a potent B cell mitogen. Here we report that CpG-A is internalized by B cells, and CpG-A and CpG-B accumulate to distinct intracellular compartments. When present in the form of an immune complex (CpG-A IC), CpG-A is taken up more efficiently by AM14 IgG2a-specific B cells, and elicits a robust TLR9-dependent B cell proliferative response. B cells proliferating comparably and in a TLR9-dependent fashion in response to CpG-A IC and CpG-B exhibited distinct cytokine profiles. CpG-A IC induced enhanced production of RANTES and markedly reduced levels of IL-6 when compared to CpG-B. We also found that engagement of the AM14 BCR by a protein IC, which cannot by itself induce proliferation, promoted TLR9-dependent but BCR-independent proliferation by bystander CpG-A or fragments of mammalian dsDNA. These data identify direct and indirect mechanisms by which BCR engagement facilitates access of exogenous ligands to TLR9-associated compartments and subsequent B cell activation.
Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive BCR transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1/RAGE interaction was important for this response . To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1/DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE+ and RAGE− AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.
HMGB1; RAGE; AM14 B cells; TLR9; Systemic Lupus Erythematosus; autoreactive B cell activation
Homomeric α7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified α9 subunit can also form functional homopentamers as well as α9α10 heteropentamers Current fluorescent probes for α7 nicotinic acetylcholine receptors are derived from α-bungarotoxin. However, α-bungarotoxin also binds to α9* and α1* receptors which are coexpressed with α7 in multiple tissues. We used an analog of α-conotoxin ArIB to develop a highly-selective fluorescent probe for α7 receptors. This fluorescent α-conotoxin, Cy3-ArIB[V11L;V16A], blocked acetylcholine-evoked α7 currents in Xenopus laevis oocytes with an IC50 value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike fluorescein isothiocyanate-conjugated α-bungarotoxin, Cy3-ArIB[V11L;V16A] did not block α9α10 or α1β1δε receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [125I]-α-bungarotoxin binding to mouse hippocampal membranes with a Ki value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labelling of KXα7R1 cells but not KXα3β2R4, KXα3β4R2, or KXα4β2R2 cells. This labelling could be abolished by pre-treatment with α-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for α7 receptors.
α7 nAChR; α-conotoxin; fluorescent; HEK293 cells
Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR−/− B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1−/− B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-β feedback loop and constitutively low expression of TLR7 in the IFNAR1−/− B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses.
Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs)6 through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a reactive B cells. By contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR-crosslinking alone is insufficient to activate low affinity autoreactive B cells. Importantly, priming B cells with IFN-α lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-α can contribute to the pathogenesis of systemic lupus erythematosus (SLE).
Autoantigens that contain DNA, RNA, or self-IgG are preferred targets for autoantibodies in Systemic Lupus Erythematosus (SLE). B cells promote SLE pathogenesis by: producing autoantibodies, activating autoreactive T cells, and secreting cytokines. We discuss how certain autoreactive B cells are selectively activated, with emphasis on the roles of key Toll-like receptors (TLRs). Although TLR7, which recognizes ssRNA, promotes autoimmune disease, TLR9, which recognizes DNA, unexpectedly regulates disease, despite being required for the secretion of anti-chromatin autoantibodies. We describe positive feedback loops involving B cells, T cells, DCs and soluble mediators and how these networks are regulated by TLR signals.
Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603–607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837–847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-α, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.
Systemic autoimmune disease in humans and mice is characterized by loss of immunologic tolerance to a restricted set of self-nuclear antigens. Autoantigens, such as double-stranded (ds) DNA and the RNA-containing Smith antigen (Sm), may be selectively targeted in systemic lupus erythematosus because of their ability to activate a putative common receptor. Toll-like receptor 9 (TLR9), a receptor for CpG DNA, has been implicated in the activation of autoreactive B cells in vitro, but its role in promoting autoantibody production and disease in vivo has not been determined. We show that in TLR9-deficient lupus-prone mice, the generation of anti-dsDNA and antichromatin autoantibodies is specifically inhibited. Other autoantibodies, such as anti-Sm, are maintained and even increased in TLR9-deficient mice. In contrast, ablation of TLR3, a receptor for dsRNA, did not inhibit the formation of autoantibodies to either RNA- or DNA-containing antigens. Surprisingly, we found that despite the lack of anti-dsDNA autoantibodies in TLR9-deficient mice, there was no effect on the development of clinical autoimmune disease or nephritis. These results demonstrate a specific requirement for TLR9 in autoantibody formation in vivo and indicate a critical role for innate immune activation in autoimmunity.