C-terminal citrullinated peptide alters antigen-specific APC:T cell interactions leading to breach of immune tolerance.

In rheumatoid arthritis, the emergence of anti-citrullinated autoimmunity is associated with HLA-antigen-T cell receptor complexes. The precise mechanisms underpinning this breach of tolerance are not well understood. Porphyromonas gingivalis expresses an enzyme capable of non-endogenous C-terminal citrullination with potential to generate citrullinated autoantigens. Here we document how C-terminal citrullination of ovalbumin peptide323-339 alters the interaction between antigen-presenting cells and OTII T cells to induce functional changes in responding T cells. These data reveal that C-terminal citrullination is sufficient to breach T cell peripheral tolerance in vivo and reveal the potential of C-terminal citrullination to lower the threshold for T cell activation. Finally, we demonstrate a role for the IL-2/STAT5/CD25 signalling axis in breach of tolerance. Together, our data identify a tractable mechanism and targetable pathways underpinning breach of tolerance in rheumatoid arthritis and provide new conceptual insight into the origins of anti-citrullinated autoimmunity.

Immunoglobulin A antibodies to oxidized collagen type II as a potential biomarker for the stratification of spondyloarthritis from rheumatoid arthritis.

OBJECTIVES: The discovery of diseased tissue-specific neoantigens offers the opportunity to develop important disease tissue-specific biomarkers that can help in the prediction, diagnosis, and stratification of diseases. This opportunity is specifically significant for autoimmune diseases where diagnostic biomarkers are not available. Inflammatory autoimmune diseases are commonly associated with local generation of large amounts of reactive oxidants. We have previously identified oxidative post-translationally modified (oxPTM) tissue-specific neoantigens in rheumatoid arthritis (RA) and type 1 diabetes that elicit an immune response. In the current study, we studied the presence and clinical significance of antibodies to oxPTM collagen type II (CII) in patients with spondyloarthritis (SpA). METHOD: Levels of antibodies specific to native CII and oxPTM-CII were assessed by enzyme-linked immunosorbent assay. RESULTS: Immunoglobulin G (IgG) binding to oxPTM-CII was observed in 52%, 83%, and 28% of serum samples from patients with axial spondyloarthritis (axSpA), RA, and psoriatic arthritis (PsA), respectively. Importantly, while strong IgA anti-oxPTM-CII responses were detected in axSpA and PsA patients, with 47% and 84% respective binders, no IgA anti-oxPTM-CII was detected in RA patients. IgA anti-oxPTM-CII reactivity in axSpA patients treated with biologics was higher and more frequent, with 85% binders compared to 9% binders in patients treated with synthetic disease-modifying anti-rheumatic drugs. CONCLUSION: Our data imply that SpA and PsA are associated with the presence of antibodies to oxPTM-CII, suggesting that there may be a humoral component that may distinguish patients with SpA from RA. Our approach could be adapted to other diseases, particularly to inflammatory autoimmune diseases.

Accelerated post traumatic osteoarthritis in a dual injury murine model.

OBJECTIVE: Joint injury involving destabilisation of the joint and damage to the articular cartilage (e.g., sports-related injury) can result in accelerated post-traumatic osteoarthritis (PTOA). Destabilised medial meniscotibial ligament (DMM) surgery is one of the most commonly used murine models and whilst it recapitulates Osteoarthritis (OA) pathology, it does not necessarily result in multi-tissue injury, as occurs in PTOA. We hypothesised that simultaneous cartilage damage and joint destabilisation would accelerate the onset of OA pathology. METHODS: OA was induced in C57BL/6 mice via (a) DMM, (b) microblade scratches of articular cartilage (CS) or (c) combined DMM and cartilage scratch (DCS). Mice were culled 7, 14 and 28 days post-surgery. Microcomputed tomography (µCT) and histology were used to monitor bone changes and inflammation. Dynamic weight bearing, an indirect measure of pain, was assessed on day 14. RESULTS: Osteophytogenesis analysis via µCT revealed that osteophytes were present in all groups at days 7 and 14 post-surgery. However, in DCS, osteophytes were visually larger and more numerous when compared with DMM and cartilage scratch (CS). Histological assessment of cartilage at day 14 and 28, revealed significantly greater damage in DCS compared with DMM and CS. Furthermore, a significant increase in synovitis was observed in DCS. Finally, at day 14 osteophyte numbers correlated with changes in dynamic weight bearing. CONCLUSION: Joint destabilisation when combined with simultaneous cartilage injury accelerates joint deterioration, as seen in PTOA. Thus, DCS provides a novel and robust model for investigating multiple pathological hallmarks, including osteophytogenesis, cartilage damage, synovitis and OA-related pain.

Antibodies to heteromeric glycolipid complexes in multifocal motor neuropathy.

BACKGROUND: Measurement of anti-GM1 IgM antibodies in multifocal motor neuropathy (MMN) sera is confounded by relatively low sensitivity that limits clinical usefulness. Combinatorial assay methods, in which antibodies react to heteromeric complexes of two or more glycolipids, are being increasingly applied to this area of diagnostic testing. METHODS: A newly developed combinatorial glycoarray able to identify antibodies to 45 different heteromeric glycolipid complexes and their 10 individual glycolipid components was applied to a randomly selected population of 33 MMN cases and 57 normal or disease controls. Comparison with an enzyme-linked immunosorbent assay (ELISA) was conducted for selected single glycolipids and their complexes. RESULTS: By ELISA, 22/33 MMN cases had detectable anti-GM1 IgM antibodies, whereas 19/33 MMN samples were positive for anti-GM1 antibodies by glycoarray. Analysis of variance (anova) revealed that of the 55 possible single glycolipids and their 1:1 complexes, antibodies to the GM1:galactocerebroside (GM1:GalC) complex were most significantly associated with MMN, returning 33/33 MMN samples as positive by glycoarray and 29/33 positive by ELISA. Regression analysis revealed a high correlation in absolute values between ELISA and glycoarray. Receiver operator characteristic analysis revealed insignificantly different diagnostic performance between the two methods. However, the glycoarray appeared to offer slightly improved sensitivity by identifying antibodies in four ELISA-negative samples. CONCLUSIONS: The use of combinatorial glycoarray or ELISA increased the diagnostic sensitivity of anti-glycolipid antibody testing in this cohort of MMN cases, without significantly affecting specificity, and may be a useful assay modification for routine clinical screening.

Moderate exercise protects against joint disease in a murine model of osteoarthritis.

Exercise is recommended as a non-pharmacological therapy for osteoarthritis (OA). Various exercise regimes, with differing intensities and duration, have been used in a range of OA rodent models. These studies show gentle or moderate exercise reduces the severity of OA parameters while high intensity load bearing exercise is detrimental. However, these studies were largely conducted in rats or in mouse models induced by severe injury, age or obesity, whilst destabilization of the medial meniscus (DMM) in mice has become a widely accepted model due to its lower variability, moderate progression and timescale. The present study was undertaken to provide insight into the effect of moderate exercise on early joint pathology in the DMM mouse model. Exercise was induced a week after induction by forced wheel walking for three or 7 weeks. Joints were analyzed by microcomputed tomography and histology. Assessment of skeletal parameters revealed that exercise offered protection against cartilage damage after 7 weeks of exercise, and a temporary protection against osteosclerosis was displayed after 3 weeks of exercise. Furthermore, exercise modified the metaphyseal trabecular microarchitecture of the osteoarthritic leg in both time points examined. Collectively, our findings corroborate previous studies showing that exercise has an important effect on bone in OA, which subsequently, at 8 weeks post-induction, translates into less cartilage damage. Thus, providing an exercise protocol in a surgical mouse model of OA, which can be used in the future to further dissect the mechanisms by which moderate exercise ameliorates OA.

Therapeutic glucocorticoids prevent bone loss but drive muscle wasting when administered in chronic polyarthritis.

BACKGROUND: Patients with rheumatoid arthritis (RA) experience extra-articular manifestations including osteoporosis and muscle wasting, which closely associate with severity of disease. Whilst therapeutic glucocorticoids (GCs) reduce inflammation in RA, their actions on muscle and bone metabolism in the context of chronic inflammation remain unclear. We utilised the TNF-tg model of chronic polyarthritis to ascertain the impact of therapeutic GCs on bone and muscle homeostasis in the context of systemic inflammation. METHODS: TNF-tg and wild-type (WT) animals received either vehicle or the GC corticosterone (100 µg/ml) in drinking water at onset of arthritis. Arthritis severity and clinical parameters were measured, serum collected for ELISA and muscle and bone biopsies collected for µCT, histology and mRNA analysis. In vivo findings were examined in primary cultures of osteoblasts, osteoclasts and myotubes. RESULTS: TNF-tg mice receiving GCs showed protection from inflammatory bone loss, characterised by a reduction in serum markers of bone resorption, osteoclast numbers and osteoclast activity. In contrast, muscle wasting was markedly increased in WT and TNF-tg animals receiving GCs, independently of inflammation. This was characterised by a reduction in muscle weight and fibre size, and an induction in anti-anabolic and catabolic signalling. CONCLUSIONS: This study demonstrates that when given in early onset chronic polyarthritis, oral GCs partially protect against inflammatory bone loss, but induce marked muscle wasting. These results suggest that in patients with inflammatory arthritis receiving GCs, the development of interventions to manage deleterious side effects in muscle should be prioritised.

Genetic imprinting of autoantibody repertoires in systemic lupus erythematosus patients.

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.

Monoclonal antibodies raised against Guillain-Barré syndrome-associated Campylobacter jejuni lipopolysaccharides react with neuronal gangliosides and paralyze muscle-nerve preparations.

Guillain-Barré 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.

Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins.

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 V(H) and V(L) 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.

Anti-GQ1b antibodies and evoked acetylcholine release at mouse motor endplates.

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.

Anti-GQ1b ganglioside antibodies mediate complement-dependent destruction of the motor nerve terminal.

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.