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Objective Patients with rheumatoid arthritis (RA) are at increased risk of developing cardiovascular disease (CVD) via mechanisms that have not yet been defined. Inflammatory pathways, in particular within the vascular adventitia, are implicated in the pathogenesis of primary CVD but could be amplified in RA at the local tissue level. The aim of this study was to examine the aortic adventitia of coronary artery disease (CAD) patients with or without RA to determine the cytokine profile contained therein. Methods Aortic adventitia and internal thoracic artery biopsy specimens obtained from 19 RA patients and 20 non‐RA patients undergoing coronary artery bypass graft surgery were examined by immunohistochemistry. Results Interleukin‐18 (IL‐18), IL‐33, and tumor necrosis factor (TNF) were expressed in aortic adventitia biopsy specimens from both groups, and expression of these cytokines was significantly higher in RA patients. In RA patients, IL‐33 expression in endothelial cells correlated positively with the number of swollen joints, suggesting a link between the systemic disease state and the local vascular tissue microlesion. Conclusion The presence of the proinflammatory cytokines IL‐18, IL‐33, and TNF may play a role in the inflammatory process within the adventitia that contributes to plaque formation and destabilization. In theory, the amplified expression of these cytokines may contribute to the known increased occurrence and severity of CAD in patients with RA.

The advent of a variety of cytokine inhibitors in the treatment of autoimmune and autoinflammatory diseases has transformed the clinical outlook in recent years. However, a proportion of patients either does not respond or exhibits only partial or short-lived benefits. As such, there remains intense interest in identifying new pathways that can yield therapeutic benefit when modulated. The IL-17 pathway has been identified in murine models as a potent driver of autoimmune phenomena. IL-17 family cytokines are released by a several leukocyte subsets, primarily T cells and innate lymphocytes. In turn, this family is induced by a variety of upstream cytokines, including IL-23. Several clinical trials have now identified marked benefits upon inhibition of the IL-17/IL-23 axis, particularly in psoriasis. Such data are being extended in a variety of clinical inflammatory diseases with variable benefit arising, including potential deficit. It is likely that agents that target IL-17 and IL-23 will find a place in the management of psoriasis initially with other disease states emerging over time.

The effective functioning of the adaptive immune system requires careful clonal regulation within the B cell compartment. Some microbial pathogens produce virulence factors, like staphylococcal protein A, which interact at high frequencies with B lymphocyte through unconventional binding sites in BCR variable region frameworks conserved during evolution. We have characterized the in vivo effect of staphylococcal protein A treatment on peripheral B cells bearing susceptible BCR, and found a dose-dependent direct relationship over the range of 2 mg to

Summary Background Monoclonal antibodies are a valuable tool in the study of allergens, but the technology used in their generation can be slow and labour-intensive. Therefore, we have examined recombinant antibody development by phage-display against single allergens and protein mixtures. Objective We used the avian immunoglobulin system (generated from single VH and VL genes) to provide a rapid method for generating highly specific recombinant antibody fragments from a minimal number of animals. Methods A single-chain antibody fragment (scFv) library was generated from a single chicken immunized with model allergens. ScFvs were isolated by phage-display and their properties investigated by ELISA and Western blot. Results Mono-specific scFvs were generated against recombinant Fel d 1 and native Amb a 1. Pannings against yellow jacket venom extracts only yielded clones that reacted with multiple proteins in the venom extract. The scFvs from each panning type were effectively expressed in Escherichia coli and readily purified. Highly specific and sensitive recognition of Fel d 1 and Amb a 1 was demonstrated in ELISA, with scFvs displaying antibody-concentration-dependent absorbance curves down to picogram levels of antibody. The specificity of selected antibodies for their cognate antigen was further confirmed in Western blot analysis, with scFvs directed to either Fel d 1 or Amb a 1 showing no reactivity for the other antigens used in immunization. Anti-Amb a 1 scFvs also mapped Amb a 1-isoform location in Western blot of ragweed extracts separated by 2D SDS-PAGE. DNA sequence analysis of scFvs showed that multiple different clones had been generated against Fel d 1 and Amb a 1. Using two anti-Fel d 1 scFv for ELISA analysis of Fel d 1 content in crude cat pelt extracts, we could produce data which were highly similar (P=0.33 and 0.89 by paired t-test analysis) to those obtained using conventional assays (radial immunodiffusion). Conclusion Phage-display technology may generate multiple allergen-specific recombinant antibody fragments from a single chicken, to allergens from mammalian, plant and insect sources. The resulting antibody fragments are of demonstrable use in allergen identification and quantification, in comparison with standard immunoassays.

Mutations in the human CIAS1 (hCIAS1) gene have been identified in a continuum of inflammatory disorders including familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal onset multisystem inflammatory disease (NOMID). CIAS1 codes for the protein Cryopyrin, which appears to play a role in innate immune function by regulating the production of proinflammatory cytokines. Human and mouse Cryopyrin are highly conserved and consist of three functional domains including a pyrin domain, an NACHT domain, and a leucine-rich repeat (LRR) domain that are characteristics of the NALP family of proteins. The pyrin and NACHT domains of Cryopyrin and other NALP proteins are highly conserved among primate and nonprimate mammals, suggesting purifying selection throughout mammalian evolution. Cryopyrin expression is also very similar in human and mouse with mouse CIAS1 mRNA expression found primarily in peripheral blood leukocytes consistent with the postulated inflammatory function. We also detected significant expression in mouse eye and skin tissue, which is consistent with symptoms observed in human Cryopyrin-associated diseases.

Contributing to host defenses from the adaptive immune system, splenic marginal zone (MZ) B cells, with their preactivated state and special topographical location, serve essential roles as primary defenders from blood-borne microbes. From studies designed to define the immunologic impact of protein A ofStaphylococcus aureus(SpA), a virulence factor with targeted B cell antigen receptor-binding properties, we found that within minutes ofin vivoexposure, SpA became surface associated with B lymphocytes and induced trafficking. Within several hours, MZ were completely effaced of affected B cells. This was rapidly followed by massive B cell apoptosis, with accelerated preferential deletion of targeted MZ B cells and impaired responsiveness to T independent immunogens. Subsequently, the temporal recovery of MZ B cells was significantly delayed compared to peripheral follicular B cells (B-2 cells). These studies elucidate the cellular program induced by a natural toxin that is shown to be highly efficient at depleting innate-like B cells important for defense from systemic infection.

Abs specific for phosphorylcholine (PC) are known to contribute to the immune defense against a variety of microbial infections. To assess for other types of binding interactions, we performed surveys of anti-PC Abs of diverse biologic origins and structural diversity and demonstrated a common autoreactivity for oxidatively modified low density lipoprotein and other oxidation-specific structures containing PC-Ags. We also found that cells undergoing apoptosis sequentially express a range of oxidation-specific neo-self PC determinants. Whereas natural Abs to PC recognized cells at early stages of apoptosis, by contrast, an IgG anti-PC Ab, representative of a T cell-dependent response, recognized PC determinants primarily associated with late stages of apoptosis. Cumulatively, these results demonstrate a fundamental paradigm in which Abs from both the innate and the T cell-dependent tiers of the B cell compartment recognize a minimal molecular motif arrayed both on microbes and as neo-self Ags linked to atherosclerosis and autoimmune disease.

During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low-density lipoprotein (oxLDL) and active immunization of hypercholesterolemic mice with oxLDL ameliorates atherogenesis. We unexpectedly found that many autoantibodies to oxLDL derived from 'naive' atherosclerotic mice share complete genetic and structural identity with antibodies from the classic anti-phosphorylcholine B-cell clone, T15, which protect against common infectious pathogens, including pneumococci. To investigate whether in vivo exposure to pneumococci can affect atherogenesis, we immunized Ldlr(-/-) mice with Streptococcus pneumoniae. This induced high circulating levels of oxLDL-specific IgM and a persistent expansion of oxLDL-specific T15 IgM-secreting B cells primarily in the spleen, which were cross-reactive with pneumococcal determinants. Pneumococcal immunization decreased the extent of atherosclerosis, and plasma from these mice had an enhanced capacity to block the binding of oxLDL to macrophages. These studies show molecular mimicry between epitopes of oxLDL and S. pneumoniae and indicate that these immune responses can have beneficial effects.

Amongst the many ploys used by microbial pathogens to interfere with host immune responses is the production of proteins with the properties of superantigens. These properties enable superantigens to interact with conserved variable region framework subdomains of the antigen receptors of lymphocytes rather than the complementarity determining region involved in the binding of conventional antigens. To understand how a B cell superantigen affects the host immune system, we infused protein A of Staphylococcus aureus (SpA) and followed the fate of peripheral B cells expressing B cell receptors (BCRs) with VH regions capable of binding SpA. Within hours, a sequence of events was initiated in SpA-binding splenic B cells, with rapid down-regulation of BCRs and coreceptors, CD19 and CD21, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, and DNA fragmentation. After exposure, B cell apoptotic bodies were deposited in the spleen, lymph nodes, and Peyer's patches. Although in vivo apoptosis did not require the Fas death receptor, B cells were protected by interleukin (IL)-4 or CD40L, or overexpression of Bcl-2. These studies define a pathway for BCR-mediated programmed cell death that is VH region targeted by a superantigen.

The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and sometimes esoteric instrument, the flow cytometer that is able to count hundreds of cells in a single second, and can provide repetitive results in a tireless manner. Given this, the possibility to analyse immune phenotypes in a variety of clinical conditions has changed the use of the flow cytometer, which was incidentally invented in the late 1960s to measure cellular DNA by using intercalating dyes, such as ethidium bromide. The epidemics of HIV/AIDS in the 1980s then gave a dramatic impulse to the technology of counting specific cells, since it became clear that the quantification of the number of peripheral blood CD4+ T cells was crucial to follow the course of the infection, and eventually for monitoring the therapy. As a consequence, the development of flow cytometers that had to be easy-to-use in all clinical laboratories helped to widely disseminate this technology. Nowadays, it is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions. Of note, recent developments have created the sophisticated technology of mass cytometry, which is able to simultaneously identify dozens of molecules at the single cell level and allows us to better understand the complexity and beauty of the immune system.

Multiple myeloma (MM) is closely associated with bone destruction. Once migrated to the bone marrow, MM cells unbalance bone formation and resorption via the recruitment and maturation of osteoclast precursors. The Notch pathway plays a key role in different types of cancer and drives several biological processes relevant in MM, including cell localization within the bone marrow, proliferation, survival and pharmacological resistance. Here we present evidences that MM can efficiently drive osteoclastogenesis by contemporaneously activating Notch signaling on tumor cells and osteoclasts through the aberrant expression of Notch ligands belonging to the Jagged family. Active Notch signaling in MM cells induces the secretion of the key osteoclastogenic factor, RANKL, which can be boosted in the presence of stromal cells. In turn, MM cells-derived RANKL causes the upregulation of its receptor, RANK, and Notch2 in pre-osteoclasts. Notch2 stimulates osteoclast differentiation by promoting autocrine RANKL signaling. Finally, MM cells through Jagged ligands expression can also activate Notch signaling in pre-osteoclast by direct contact. Such synergism between tumor cells and pre-osteoclasts in MM-induced osteoclastogenesis can be disrupted by silencing tumor-derived Jagged1 and 2. These results make the Jagged ligands new promising therapeutic targets in MM to contrast bone disease and the associated co-morbidities.

The incidence of atherosclerosis is significantly increased in rheumatoid arthritis (RA). Infection is one factor that may be involved in the pathogenesis of both diseases. The cause of RA and atherosclerosis is unknown, and infection is one of the factors that may be involved in the pathogenesis of both diseases. The aims of this study were to identify bacteria in the aortic adventitia of patients with cardiovascular disease (CVD) in the presence and absence of RA, and to determine the effect of identified candidate pathogens on Toll-like receptor (TLR)-dependent signalling and the proinflammatory response. The aortic adventitia of 11 CVD patients with RA (RA+CVD) and 11 CVD patients without RA (CVD) were collected during coronary artery bypass graft surgery. Bacteria were detected in four samples from CVD patients and three samples from RA+CVD patients and identified by 16S rRNA gene sequencing. Methylobacterium oryzae was identified in all three RA+CVD samples, representing 44.1% of the bacterial flora. The effect of M. oryzae on TLR-dependent signalling was determined by transfection of HEK-293 cells. Although mild TLR2 signalling was observed, TLR4 was insensitive to M. oryzae. Human primary macrophages were infected with M. oryzae, and a TLDA qPCR array targeting 90 genes involved in inflammation and immune regulation was used to profile the transcriptional response. A significant proinflammatory response was observed, with many of the up-regulated genes encoding proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α) and chemokines (CCR7, IL-8). The aortic adventitia of CVD patients contains a wide range of bacterial species, and the bacterial flora is significantly less diverse in RA+CVD than CVD patients. M. oryzae may stimulate an proinflammatory response that may aggravate and perpetuate the pathological processes underlying atherosclerosis in RA patients.

Miller Fisher syndrome (MFS) is clinically characterized by ataxia, areflexia, and ophthalmoplegia, and is associated with serum anti-GQ1b-ganglioside antibodies. We have previously shown that anti-GQ1b antibodies induce complement-dependent, alpha-latrotoxin-like effects at mouse neuromuscular junctions (NMJs) in vitro. This effect comprises a massive increase in spontaneous quantal acetylcholine (ACh) release, accompanied by block of evoked release and muscle paralysis. This mechanism may contribute to the motor features of MFS. Whether the block of evoked ACh release is a primary effect of anti-GQ1b antibodies or occurs secondary to massive complement-dependent spontaneous release is unknown. Using conventional micro-electrode methods, we measured in detail ACh release evoked with low- and high-rate nerve stimulation, and studied the effect on it of a purified MFS IgG and a mouse monoclonal anti-GQ1b IgM (without added complement). We found that evoked transmitter release was unaffected. Control experiments proved binding of anti-GQ1b antibody at the NMJ. We conclude that the block of nerve-evoked ACh release at the NMJ is not a primary effect of anti-GQ1b antibodies, but is dependent on antibody-mediated complement activation. It remains to be determined whether the block of nerve-evoked ACh release is the consequence of massive spontaneous ACh release or occurs as a concomitant event.

Autoantibodies to glycans present on glycolipids mediate the postinfectious paralytic disease, Guillain–Barre syndrome (GBS). These glycans are also found on lipo-oligosaccharides (LOSs) of GBS-inducing microbes, suggesting molecular mimicry as a mechanism for disease induction. How B lymphocyte tolerance to self-glycans is regulated during the initiation phase of the disease is currently under investigation. The discovery of antiglycolipid antibodies that bind to heteromeric glycolipid complexes has generated new insights in this field. Heteromeric complexes are structurally distinct glycolipids that interact to form new molecular shapes capable of either enhancing or attenuating recognition by autoantibodies. Although the principles emerging from this phenomenon have a substantial impact on diagnostics methods, they also raise intriguing questions about the diversity of innate antibody repertoires, mechanisms of tolerance, and autoantibody targeting of neural membranes.

Background Extracellular vesicles (EVs) like microvesicles (MVs) and exosomes (EXOs) are released in an evolutionary conserved manner by cells. EVs mediate intercellular communication with the transmission of molecules from their parent cell to their targets. Objectives Our objective was to investigate the effect of EVs on human in vitro osteoclastogenesis in healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected. EVs were isolated by filtration and differential centrifugation. CD14+ cells were isolated from PBMCs by using positive selection, and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs. Then the samples were treated with 50 ng/ml recombinant human RANKL and blood-derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartarate resistant acid phosphatase (TRAP). TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results Healthy and RA-derived exosomes significantly (p Conclusions Our data suggest that EXOs profoundly regulate human osteoclastogenesis. PsA-derived EXOs stimulate, while healthy and RA-derived EXOs inhibit the osteoclast differentiation. Disclosure of Interest None declared

Introduction Extracellular vesicles (EV) are subcellular sized intercellular messengers, which are present in various biological fluids. EVs carry a wide variety of biomolecules and they may alter the recipient cells’ functions. The microvesicles are plasma membrane derived EVs; the exosomes, the smallest vesicles, originate from the endosome. Our objectives were to investigate the effect of EVs on the human in vitro osteoclastogenesis, and to characterise the serum EV profile of healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected into acid citrate dextrose vacutainer tubes. EVs were isolated by filtration and centrifugation. EV samples were stained with fluorescent anti-CD3, CD14, CD15, CD19, CD42b, CD235a antibodies (BioLegend) and detected by flow cytometry. CD14 + cells were extracted from PBMCs by using positive selection method (StemCell), and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs (PeproTech). Then the samples were treated with 50–50 ng/ml recombinant human M-CSF and RANKL (PeproTech) and with or without blood derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartrate resistant acid phosphatase (TRAP) using a commercially available kit (Sigma) . TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results RA and PsA EV samples showed increased expression of lymphocyte markers (CD3, CD19) compared to healthy donor-derived EVs (n = 6). Microvesicles did not alter the number of mature osteoclasts. By contrast, exosomes significantly (p Conclusion Our present data suggest that the plasma profile of EVs might be altered in active arthritis compared to physiological condition, and exosomes or their cargo can regulate the human osteoclastogenesis.

Background and objectives Destabilisation of the medial meniscus (DMM) and section of the anterior cruciate ligament (ACL) are commonly used in vivo models of osteoarthritis (OA). Using explant culture, the secretory profile of synovium and patellar ligament was evaluated in DMM and ACL models. Material and methods All murine procedures were Home Office (UK) approved. OA was induced in 10–12 week old C57BL/6 male mice by either DMM or ACL (1). SHAM and naive animals were used as controls. Knee joint diameter (KJD) was assessed prior to induction of arthritis and every three days thereafter. At 4 and 12 weeks post-surgery, synovium and patellar ligament were harvested and used for explant culture. Supernatants were collected after 4 h and analysed by Luminex. In parallel experiments, joints were collected for histological analysis ofF4/80+, IL-33+ and CCR4+ cells in synovium and ligaments. Freund’s complete adjuvant (FCA) monoarthritis model was used as a positive control of inflammatory joint disease. Data were expressed as mean values ±SEM (n = 6) and analysed using a 1 or 2 way ANOVA, with p Results At 4 weeks post-surgery no change in KJD was observed in either DMM or ACL. Nonetheless, secretion of MIP-1β/CCL4 was significantly increased in explant cultures from DMM and ACL compared to naive controls (p Conclusion This study characterised the secretory profile of DMM and ACL joint tissue at 4 and 12 weeks post-surgery. This profile alters with time, with reduced IL-33 and an absence of CCL4 at 12 weeks. The potential role of IL-33 and CCL4 in the early and late stage pathogenesis of these OA models merits further investigation. Reference Glasson SS, et al. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage. 2007;15(9):1061–1069

Background and objectives GM-CSF is a pivotal growth factor/cytokine that can drive aspects of RAimmunopathogenesis. In particular, GM-CSF can activate monocytes and induce their differentiation towards alternative phenotypes. The synovial environment also contains additional factors that can influence monocyte activation such as damage associated molecular patterns (DAMPs); many of which signal via TLR4. However, the impact of GM-CSF on monocytes in a synovial setting is not well understood and therefore we aimed to investigate the response of monocytes to GM-CSF in the context of synovial fluid (SF) co-stimulation. Materials and methods Monocytes isolated from human blood or murine bone marrow were cultured in the presence or absence of GM-CSF (1ng/ml) and co-stimulated with SF (2.5%/5%) from RA or OA patients. Supernatants were harvested after 24 h and chemokine and cytokine secretion was analysed by ELISA and/or MSD. Immune complexes were made by incubating human IgG and Staphylococcal Protein A at a ratio of 4:2 respectively. Results Monocytes from healthy individuals and RA patients, secreted substantial amounts of CCL17 upon stimulation with GM-CSF. In healthy monocytes, GM-CSF driven induction of CCL17 was significantly inhibited (P Conclusions GM-CSF induced production of CCL17 in monocytes is inhibited by RA-SF. The underlying mechanism is not known, however, our work shows that it is TLR-independent and downstream of STAT5 phosphorylation. Immune complexes also inhibit GM-CSF induction of CCL17 suggesting these may contribute to the phenomenon.

Background:\ud \ud Guillain-Barré syndrome (GBS) is a post-infectious polyradiculoneuropathy, frequently associated with antecedent Campylobacter jejuni (C. jejuni) infection. The presence of sialic acid on C. jejuni lipo-oligosaccharide (LOS) is considered a risk factor for development of GBS as it crucially determines the structural homology between LOS and gangliosides, explaining the induction of cross-reactive neurotoxic antibodies. Sialylated C. jejuni are recognised by TLR4 and sialoadhesin; however, the functional implications of these interactions in vivo are unknown.\ud \ud Methodology/Principal Findings:\ud \ud In this study we investigated the effects of bacterial sialylation on phagocytosis and cytokine secretion by mouse myeloid cells in vitro and in vivo. Using fluorescently labelled GM1a/GD1a ganglioside-mimicking C. jejuni strains and corresponding (Cst-II-mutant) control strains lacking sialic acid, we show that sialylated C. jejuni was more efficiently phagocytosed in vitro by BM-MΦ, but not by BM-DC. In addition, LOS sialylation increased the production of IL-10, IL-6 and IFN-β by both BM-MΦ and BM-DC. Subsequent in vivo experiments revealed that sialylation augmented the deposition of fluorescent bacteria in splenic DC, but not macrophages. In addition, sialylation significantly amplified the production of type I interferons, which was independent of pDC.\ud \ud Conclusions/Significance:\ud \ud These results identify novel immune stimulatory effects of C. jejuni sialylation, which may be important in inducing cross-reactive humoral responses that cause GBS

Background: Multiple Myeloma (MM) is currently incurable, with a median survival of 5-7 years post diagnosis. MM-associated bone disease (MM-OBD), represents a major cause of morbidity and mortality in MM patients. Despite current therapies for MM-OBD exhibiting significant potential (e.g. Zoledronate), their clinical use has been restricted due to severe treatment associated toxicities. Safe novel therapies for MM-OBD are therefore crucially required. Molecular crosstalk between receptor activator of nuclear factor Kappa B ligand (RANKL), present on and secreted by MM plasma cells, and its corresponding receptor (RANK) on osteoclast precursors (OCPs), represents a key mechanism driving osteoclastogenesis and subsequent bone pathology in MM. Our previous studies have demonstrated that Fc receptor (FcR)-mediated signals can inhibit RANKL induced osteoclastogenesis in vitro1. In addition, findings from preliminary studies show that FcR-mediated signalling in pre-osteoclasts can reduce MM plasma cell driven osteoclastogenesis in vitro. Further interrogation of the underlying molecular mechanisms show that FcR-mediated signals profoundly reduce RANK transcript, and subsequent protein expression, in pre-osteoclasts. However, the effects of FcR engagement on MM-OBD in vivo, and the FcR elicited signalling pathways responsible for inhibition of RANK expression have still to be elucidated. Aims: This study aimed to determine the in vivo potential of FcR engagement to treat bone disease in a pre-clinical model of MM. Additionally, the mechanisms underlyingFcR-mediated down-regulation of RANK expression in OCPs were interrogated. Methods: The well-characterised 5TGM1 murine model of MM, together with micro-computed topography (micro-CT), were used to evaluate the effect of FcR engagement on MM-OBD. FcR stimulation was achieved by I.P. injecting mice (every other day, following 5TGM1 I.V. cell injection), with 100 μg/ml of Protein A derived from Staphylococcus aureus (SpA). SpA has been shown to form small immune complexes (SICs) through its affinity for endogenous IgG, which in turn binds to FcγR1 receptors on monocytes and pre-OCs1. Additionally, the potential involvement of FcR signalling pathways in the down-regulation of RANK in healthy and MM-derived human OCPs was determined via immunoblotting and the use of signalling pathway inhibitors. Results: Twenty six days post-myeloma cell injection, micro-CT analysis of femurs revealed that mice receiving PBS (vehicle control, n=5) exhibited a significant decrease in bone morphmetric parameters consistent with bone erosion compared to non-myeloma bearing mice (n=3); trabecullar bone volume [BV/TV] = 2.673 vs. 3.449, p=0.034; trabecullar number [Tb.N] = 0.0035 vs. 0.0042, p=0.0041; trabecullar pattern factor [Tb.Pf] = 0.2329 vs. 0.2033, p=0.0393. Importantly, myeloma bearing mice (n=5) receiving SpA, were protected from MM-OBD. In human OCPs (sourced from healthy individuals and MM patients), FcR engagement substantially activated SyK, MEK-ERK1/2, and PI3K signaling cascades. However, inhibition of these pathways failed to restore RANK transcript levels. Discussion: These findings demonstrate novel mechanisms of RANK gene expression regulation in healthy and MM OCPs, with Fc receptors representing a potential therapeutic strategy for MM-OBD. Further studies will aim to elucidate the molecular mechanisms responsible for FcR-mediated regulation of RANK gene expression. 1. MacLellan, L. M. et al. Co-opting endogenous immunoglobulin for the regulation of inflammation and osteoclastogenesis in humans and mice. Arthritis Rheum.63, 3897-3907 (2011). Disclosures No relevant conflicts of interest to declare.

Objective Cells of the monocytic lineage play fundamental roles in the regulation of health, ranging from the initiation and resolution of inflammation to bone homeostasis. In rheumatoid arthritis (RA), the inflamed synovium exhibits characteristic infiltration of macrophages along with local osteoclast maturation, which, together, drive chronic inflammation and downstream articular destruction. The aim of this study was to explore an entirely novel route of immunoglobulin-mediated regulation, involving simultaneous suppression of the inflammatory and erosive processes in the synovium. Methods Using in vivo and in vitro studies of human cells and a murine model of RA, the ability of staphylococcal protein A (SPA) to interact with and modulate cells of the monocytic lineage was tested. In addition, the efficacy of SPA as a therapeutic agent was evaluated in murine collagen-induced arthritis (CIA). Results SPA showed a capacity to appropriate circulating IgG, by generating small immunoglobulin complexes that interacted with monocytes, macrophages, and preosteoclasts. Formation of these complexes resulted in Fcγ receptor type I–dependent polarization of macrophages to a regulatory phenotype, rendering them unresponsive to activators such as interferon-γ. The antiinflammatory complexes also had the capacity to directly inhibit differentiation of preosteoclasts into osteoclasts in humans. Moreover, administration of SPA in the early stages of disease substantially alleviated the clinical and histologic erosive features of CIA in mice. Conclusion These findings demonstrate the overarching utility of immunoglobulin complexes for the prevention and treatment of inflammatory diseases. The results shed light on the interface between immunoglobulin complex–mediated pathways, osteoclastogenesis, and associated pathologic processes. Thus, therapeutic agents designed to harness all of these properties may be an effective treatment for arthritis, by targeting both the innate inflammatory response and prodestructive pathways.

Interleukin (IL)-33, a member of the IL-1 cytokine family, is an important modulator of the immune system associated with several immune-mediated disorders. High levels of IL-33 are expressed by the central nervous system (CNS) suggesting a potential role of IL-33 in autoimmune CNS diseases. We have investigated the expression and function of IL-33 in the development of experimental autoimmune encephalomyelitis (EAE) in mice. We report here that IL-33 and its receptor ST2 (IL-33Rα) are highly expressed in spinal cord tissue, and ST2 expression is markedly increased in the spinal cords of mice with EAE. Furthermore, ST2-deficient (ST2(-/-) ) mice developed exacerbated EAE compared with wild-type (WT) mice while WT, but not ST2(-/-) EAE mice treated with IL-33 developed significantly attenuated disease. IL-33-treated mice had reduced levels of IL-17 and IFN-γ but produced increased amounts of IL-5 and IL-13. Lymph node and splenic macrophages of IL-33-treated mice showed polarization toward an alternatively activated macrophage (M2) phenotype with significantly increased frequency of MR(+) PD-L2(+) cells. Importantly, adoptive transfer of these IL-33-treated macrophages attenuated EAE development. Our data therefore demonstrate that IL-33 plays a therapeutic role in autoimmune CNS disease by switching a predominantly pathogenic Th17/Th1 response to Th2 activity, and by polarization of anti-inflammatory M2 macrophages.

Mouse innate-like B cells are a heterogeneous collection of multifunctional cells that control infection, play housekeeping roles, contribute to adaptive immunity, and suppress inflammation. We show that, among leukocytes, chemokine internalization by the D6 receptor is a unique and universal feature of all known innate-like B-cell populations and, to our knowledge, the most effective unifying marker of these cells. Moreover, we identify novel D6active B1-cell subsets, including those we term B1d, which lack CD5 and CD11b but exhibit typical B1-cell properties, including spontaneous ex vivo production of IgM, IL-10, and anti-phosphorylcholine antibody. The unprecedented opportunity to examine D6 on primary cells has allowed us to clarify its ligand specificity and show that, consistent with a scavenging role, D6 internalizes chemokines but cannot induce Ca2+ fluxes or chemotaxis. Unexpectedly, however, D6 can also suppress the function of CXCR5, a critical chemokine receptor in innate-like B-cell biology. This is associated with a reduction in B1 cells and circulating class-switched anti-phosphorylcholine antibody in D6-deficient mice. Therefore, in the present study, we identify a unifying marker of innate-like B cells, describe novel B1-cell subsets, reveal a dual role for D6, and provide the first evidence of defects in resting D6-deficient mice.

INTRODUCTIONThis method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. Specifically, we used this protocol to generate libraries of domains in which we randomized six to eight codons that define the natural VH3 specificity of SpA. In our effort, these codons were variegated using the NNK or VNS doping strategy. Toward this goal, three large (60- to 88-residue) oligonucleotides with targeted degenerate codons were designed. To facilitate construction of the composite domain library, these oligonucleotides had overlapping complementary stretches of at least 12 nucleotides. Of course, for application to other domains, the number and composition of the required oligonucleotides are dependent on the specific variant domains to be generated. This protocol details Stage 1 of the method, the generation of a phage library via overlap PCR and bacterial cloning. The initial PCR amplification requires the mixing of equimolar amounts of two oligonucleotides to form a template for the subsequent PCR.

INTRODUCTIONThis method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 2 of the method, we describe the use of streptavidin-coated magnetic beads for library selection (panning). After selection, the elution strategy used to recover desirable clones should be chosen on the basis of the type of panning strategy employed, and the properties of the ligand that has been targeted. Of the several alternatives, we have compared the common recovery strategies of low pH, trypsinization of bound libraries, and competition with soluble antigen. When considering which option to employ, it is worth knowing how this may affect the type of binder that will be recovered. Low pH will dissociate most or all binders, whether they are specific or nonspecific for the selection antigen, whereas trypsin targets the fusion area between the protein and gene III, and specifically cleaves it. As a result, for the latter, only phage that bind via a protein-protein interaction will be recovered. In contrast, soluble antigen will provide specific binders of relatively high affinity because the elution will work in a competitive manner. We recommend an empiric approach to determine which is best for specific projects.

INTRODUCTIONThis method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 3 of the method, described here, the rounds of selection are analyzed to evaluate the enrichment for binders. To do this, polyethylene glycol (PEG)-precipitated phage from each round of panning are tested in an assay, be it ELISA-based or flow cytometric. The assay should include all rounds of phage samples, from the initial unselected phage to the last round and helper phage (as a negative control). The method chosen to evaluate whole phage libraries can also be used as a system of evaluating single clones from the appropriate round. The appropriate round is the one that yields a library exhibiting the best binding characteristics (or best signal in the detection assay) associated with the protein-protein interaction. This protocol outlines a basic ELISA-based analysis strategy.

INTRODUCTIONIn recent years, phage display has evolved into a powerful tool providing opportunities to define natural protein-protein interactions and to mold novel ligand receptors. The essential advantages of phage-display approaches originate in the incorporation of the protein and genetic components into a single phage particle. By providing a direct physical link between the expressed protein and the encoding genetic information, clones with desirable functional capacities can be efficiently subjected to iterative rounds of selection, followed by amplification of the selected sublibrary. Hence, during library selection (or panning), specific phage clones are progressively enriched on the basis of their specificity and affinity for ligand. Thus, relatively rare ligand-binding clones can be rescued rapidly and efficiently from large libraries. As these expression cloning systems have matured, versatile selection methods have been reported that are based on the functional properties of displayed proteins in diverse immunochemical and biological settings. This article summarizes phage-display methodology, including relevant biology, nucleotide-doping strategies, and considerations for library design.

Although natural Abs (NAbs) are present from birth, little is known about what drives their selection and whether they have housekeeping functions. The prototypic T15-NAb, first identified because of its protective role in infection, is representative of a special type of NAb response that specifically recognizes and forms complexes with apoptotic cells and which promotes cell-corpse engulfment by phagocytes. We now show that this T15-NAb IgM-mediated clearance process is dependent on the recruitment of C1q and mannose-binding lectin, which have known immune modulatory activities that also provide “eat me” signals for enhancing phagocytosis. Further investigation revealed that the addition of T15-NAb significantly suppressed in vitro LPS-induced TNF-α and IL-6 secretion by the macrophage-like cell line, RAW264.7, as well as TLR3-, TLR4-, TLR7-, and TLR9-induced maturation and secretion of a range of proinflammatory cytokines and chemokines by bone marrow-derived conventional dendritic cells. Significantly, high doses of this B-1 cell produced NAb also suppressed in vivo TLR-induced proinflammatory responses. Although infusions of apoptotic cells also suppressed such in vivo inflammatory responses and this effect was associated with the induction of high levels of IgM antiapoptotic cell Abs, apoptotic cell treatment was not effective at suppressing such TLR responses in B cell-deficient mice. Moreover, infusions of T15-NAb also efficiently inhibited both collagen-induced arthritis and anti-collagen II Ab-mediated arthritis. These studies identify and characterize a previously unknown regulatory circuit by which a NAb product of innate-like B cells aids homeostasis by control of fundamental inflammatory pathways.

Anti-GM1 ganglioside autoantibodies are used as diagnostic markers for motor axonal peripheral neuropathies and are believed to be the primary mediators of such diseases. However, their ability to bind and exert pathogenic effects at neuronal membranes is highly inconsistent. Using human and mouse monoclonal anti-GM1 antibodies to probe the GM1-rich motor nerve terminal membrane in mice, we here show that the antigenic oligosaccharide of GM1 in the live plasma membrane is cryptic, hidden on surface domains that become buried for a proportion of anti-GM1 antibodies due to a masking effect of neighboring gangliosides. The cryptic GM1 binding domain was exposed by sialidase treatment that liberated sialic acid from masking gangliosides including GD1a or by disruption of the live membrane by freezing or fixation. This cryptic behavior was also recapitulated in solid-phase immunoassays. These data show that certain anti-GM1 antibodies exert potent complement activation-mediated neuropathogenic effects, including morphological damage at living terminal motor axons, leading to a block of synaptic transmission. This occurred only when GM1 was topologically available for antibody binding, but not when GM1 was cryptic. This revised understanding of the complexities in ganglioside membrane topology provides a mechanistic account for wide variations in the neuropathic potential of anti-GM1 antibodies.

Summary Systemic lupus erythematosus (SLE) is an autoimmune disease distinguished by great heterogeneity in clinical manifestations and autoantibody expression. While only a handful of autoantibody specificities have proved useful for clinical diagnosis, to characterize complex lupus-associated autoantibody profiles more fully we have applied proteome microarray technology. Our multiplex microarrays included control ligands and 65-autoantigens, which represent diverse nuclear and cytoplasmic antigens recognized by disease-associated and natural autoantibodies. From longitudinal surveys of unrelated SLE patients, we found that autoantibody profile patterns can be patient-specific and highly stable overtime. From profiles of 38 SLE patients that included 14 sets of SLE twins, autoantibodies to the phospholipid neo-determinants, malondialdehyde (MDA) and phosphorylcholine (PC), which are exposed on apoptotic but not healthy cells, were among the most prevalent and highly expressed. We also found that immunoglobulin M (IgM) reactivity to MDA and PC ligands had significant direct correlations with DNA-containing antigens, while such a general relationship was not found with a panel of RNA-related antigens, or for IgG-autoantibodies. Significantly, hierarchical analysis revealed co-distribution/clustering of the IgM autoantibody repertoire patterns for six of 14 twin sets, and such patterns were even more common (10 of 14) for IgG autoantibody profiles. Our findings highlight the potentially distinct roles of IgM and IgG autoantibodies, as we postulate that the direct correlations for IgM autoantibodies to DNA antigens with apoptosis-related determinants may be due to co-expression arising from common pro-homeostatic protective roles. In contrast, the sharing of IgG autoantibody fingerprints by monozygotic twins suggests that lupus IgG autoantibodies can arise in predisposed individuals in genetically determined patterns.

Autoimmune neuropathies are frequently associated with pathogenic anti-ganglioside antibodies targeting ganglioside-rich neuronal and glial membranes. The extent of injury is determined by the concentration of membrane ganglioside and thus reduction might be expected to attenuate disease. In this study, we suppressed ganglioside biosynthesis in PC12 cells with the glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin and observed reduced plasma membrane antibody binding and a major neuroprotective effect in complement-mediated lysis assays. These data demonstrate that iminosugar inhibitors, currently used to treat type 1 Gaucher disease, are also of potential value for depleting antigen and thereby suppressing tissue injury in anti-ganglioside antibody-associated neuropathy.

significantly varies between BTZ adapted and CFZ adapted MM cells and can be increased by 2 inhibition.

Background and objectives HLA-B27 expression is associated with spondyloarthropathy (SpA) and transgenic rats expressing human HLA-B27 and b2-microglobulin (B27 rats) display systemic inflammation and bone loss, which resembles human SpA. We have previously shown that these rats lack a dendritic cell population. However, the myeloid compartment has not been fully characterised. Here, we aimed to characterise monocyte and pre-osteoclast subsets in the bone marrow and blood of B27 rats, and examined the potential of different monocyte/pre-osteoclast subsets to generate mature osteoclasts. Materials and methods 14–16 week old control (B7) and B27 rats were bled and plasma CCL2 levels were measured by ELISA. Monocytes subsets were analysed and quantified in both the bone marrow and blood of B27, B7 and non-transgenic animals, by evaluation of surface markers (CD172a, CD43, and CD11b) and intracellular uptake of fluorescent M-CSF and CCL2 by flow cytometry. Monocyte populations were FACS sorted and cultured in pro-osteoclastogenic medium for 7 days to evaluate osteoclastogenic potential. Cultures were stained with tartrate-resistant acid phosphatase (TRAP) and mature osteoclasts (TRAP + and ≥3 nuclei) were quantified by light microscopy. Results A previously unidentified CD172a + CD43 lo CD11b - population of monocytes was observed in the bone marrow. These are bona fide monocytes, expressing CD115 and CCR2, as determined by uptake of fluorescent M-CSF and CCL2 respectively. Interestingly, this new monocyte population was significantly increased in B27 rats. Assessment of the osteoclastogenic potential of bone marrow monocyte subsets revealed that CD172a + CD43 lo CD11b - , but not CD172a + CD43 lo CD11b + monocytes,can differentiate into mature osteoclasts. Furthermore, although CD172a + CD43 lo CD11b - have osteoclastogenic potential, optimal osteoclastogenesis was observed only when all CD43 lo (CD172a + CD43 lo CD11b - and CD172a + CD43 lo CD11b + ) were present. No differences in osteoclastogenesis were observed between B27 and controls rats. Finally, evaluation of circulating monocytes demonstrated that all blood monocytes express CD11b and that the CD43 lo CD11b + population was increased in B27 rats. This corresponded with an increase in CCL2 plasma levels in the B27 rats. Conclusions We have identified a previously unreported CD11b - monocyte population in the bone marrow of rats, which can differentiate into mature osteoclasts. Along with the numbers of total CD43 lo monocytes, this population is significantly increased in the bone marrow and blood of rats over-expressing HLA-B27. The increase in these populations in the B27 rats may contribute to enhanced inflammation and bone loss.

Marginal zone B cells and B-1 cells have been termed innate-like B cells as they express limited repertoires that play special roles in immune defenses against common infections. These B cells are the sources of natural antibodies and are capable of highly accelerated clonal responses that help counter blood-borne infections. We have characterized a class of microbial product with highly adapted binding interactions with host immunoglobulins/B cell receptors (BCRs), which enable the targeting of large supra-clonal sets of B cells for activation-associated apoptotic death. In recent studies, we have shown that all B cells with V region-targeted BCRs are susceptible. However, compared to follicular B cells, in vivo exposure preferentially causes innate-like B cells to undergo induced death with subsequent long-lasting supra-clonal depletion and immune tolerance. Based on these properties, it is likely that B cell superantigens influence the pathogenesis of some common infections, but also may provide novel therapeutic opportunities to treat B cell neoplastic and autoimmune diseases.

To interfere with host immune responses, some microbial pathogens produce proteins with the properties of superantigens, which can interact via conserved V region framework subdomains of the Ag receptors of lymphocytes rather than the complementarity-determining region involved in the binding of conventional Ags. In recent studies, we have elucidated how a model B cell superantigen affects the host immune system by targeting a conserved VH site on the Ag receptors of B lymphocytes. To determine whether these findings represent a general paradigm, we investigated the in vivo immunobiologic properties of protein L of Peptostreptococcus magnus (PpL), a microbial Ig-binding protein specific for a V region site on Ig L chains. Our studies confirmed that PpL binding is restricted to a subset of murine Vκ-expressing B cells, and found that B cells with stronger PpL-binding activity are associated with certain B cell subsets: splenic marginal zone (CD21high CD23low), splenic CD1+, peritoneal B-1a (IgDlow CD5+), and CD21high CD24high B cells in peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches. Infusion of PpL triggered a sequence of events in B cell receptor (BCR)-targeted B cells, with rapid down-regulation of BCR, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, DNA fragmentation, and the deposition of B cell apoptotic bodies. These studies define a common pathway by which microbial toxins that target V region-associated BCR sites induce programmed cell death.

Recent reports have shown that protein A of Staphylococcus aureus (SpA) is a specific toxin for B cells by virtue of specific binding interactions with conserved sites on the V(H) region of the B-cell antigen receptor. The structural basis for these Fab-binding interactions has recently been revealed in crystallographic analyses, which have demonstrated many similarities with the interactions of T-cell superantigens. Investigations of the in vivo response to SpA have illustrated how a B-cell superantigen can be used to provide a window for examining fundamental principles that underlie the immunobiology of B lymphocytes.

Miller-Fisher syndrome is an autoimmune neuropathy characterized by ataxia, areflexia and ophthalmoplegia, and in the majority of cases the presence of high titres of anti-GQ1b ganglioside antibodies. In an ex vivo model, human and mouse anti-GQ1b antibodies have been shown previously to induce a complement-dependent alpha-latrotoxin-like effect on the murine motor endplate, i.e. they bring about massive quantal release of acetylcholine and eventually block neuromuscular transmission. Using immunofluorescence microscopy with image analysis, we show here that the late stages of this electrophysiological effect temporally coincide with the loss of heavy neurofilament (200 kDa) and type III beta-tubulin immunostaining and structural breakdown of the nerve terminal, as demonstrated by electron microscopy. Ultrastructurally, axon terminals were disorganized, depleted of vesicles, and subdivided by the infiltrating processes of capping Schwann cells. These findings provide clear pathological evidence to support a role for anti-ganglioside antibodies in mediating nerve terminal injury and further advance the view that this site may be of importance as a target in some human neuropathies.

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Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients.

INTRODUCTIONThis method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 2 of the method, we describe the use of streptavidin-coated magnetic beads for library selection (panning). After selection, the elution strategy used to recover desirable clones should be chosen on the basis of the type of panning strategy employed, and the properties of the ligand that has been targeted. Of the several alternatives, we have compared the common recovery strategies of low pH, trypsinization of bound libraries, and competition with soluble antigen. When considering which option to employ, it is worth knowing how this may affect the type of binder that will be recovered. Low pH will dissociate most or all binders, whether they are specific or nonspecific for the selection antigen, whereas trypsin targets the fusion area between the protein and gene III, and specifically cleaves it. As a result, for the latter, only phage that bind via a protein-protein interaction will be recovered. In contrast, soluble antigen will provide specific binders of relatively high affinity because the elution will work in a competitive manner. We recommend an empiric approach to determine which is best for specific projects.

Guillain-Barre syndrome and its variant, Miller-Fisher syndrome, are acute, postinfectious, autoimmune neuropathies that frequently follow Campylobacter jejuni enteritis. The pathogenesis is believed to involve molecular mimicry between sialylated epitopes on C. jejuni LPSs and neural gangliosides. More than 90% of Miller-Fisher syndrome cases have serum anti-GQ1b and anti-GT1a ganglioside antibodies that may also react with other disialylated gangliosides including GD3 and GD1b. Structural studies on LPS from neuropathy-associated C. jejuni strains have revealed GT1a-like and GD3-like core oligosaccharides. To determine whether this structural mimicry results in pathogenic autoantibodies, we immunized mice with GT1a/GD3-like C. jejuni LPS and then cloned mAb’s that reacted with both the immunizing LPS and GQ1b/GT1a/GD3 gangliosides. Immunohistology demonstrated antibody binding to ganglioside-rich sites including motor nerve terminals. In ex vivo electrophysiological studies of nerve terminal function, application of antibodies either ex vivo or in vivo via passive immunization induced massive quantal release of acetylcholine, followed by neurotransmission block. This effect was complement-dependent and associated with extensive deposits of IgM and C3c at nerve terminals. These data provide strong support for the molecular mimicry hypothesis as a mechanism for the induction of cross-reactive pathogenic anti-ganglioside/LPS antibodies in postinfectious neuropathies.

ObjectivesAn interferon (IFN) gene signature (IGS) is present in approximately 50% of early, treatment naive rheumatoid arthritis (eRA) patients where it has been shown to negatively impact initial response to treatment. We wished to validate this effect and explore potential mechanisms of action.MethodsIn a multicentre inception cohort of eRA patients (n=191), we examined the whole blood IGS (MxA, IFI44L, OAS1, IFI6, ISG15) with reference to circulating IFN proteins, clinical outcomes and epigenetic influences on circulating CD19+ B and CD4+ T lymphocytes.ResultsWe reproduced our previous findings demonstrating a raised baseline IGS. We additionally showed, for the first time, that the IGS in eRA reflects circulating IFN-α protein. Paired longitudinal analysis demonstrated a significant reduction between baseline and 6-month IGS and IFN-α levels (pPARP9, STAT1,andEPSTI1, associated with baseline IGS/IFNα levels. Differentially methylated CPG sites implicated altered transcription factor binding in B cells (GATA3, ETSI, NFATC2, EZH2) and T cells (p300, HIF1α).ConclusionsOur data suggest that, in eRA, IFN-α can cause a sustained, epigenetically mediated, pathogenic increase in lymphocyte activation and proliferation, and that the IGS is, therefore, a robust prognostic biomarker. Its persistent harmful effects provide a rationale for the initial therapeutic targeting of IFN-α in selected patients with eRA.