A novel mouse model of cerebral adrenoleukodystrophy highlights NLRP3 activity in lesion pathogenesis.

Objective: We sought to create and characterize a mouse model of the inflammatory, cerebral demyelinating phenotype of X-linked adrenoleukodystrophy (ALD) that would facilitate the study of disease pathogenesis and therapy development. We also sought to cross-validate potential therapeutic targets such as fibrin, oxidative stress, and the NLRP3 inflammasome, in post-mortem human and murine brain tissues. Background: ALD is caused by mutations in the gene ABCD1 encoding a peroxisomal transporter. More than half of males with an ABCD1 mutation develop the cerebral phenotype (cALD). Incomplete penetrance and absence of a genotype-phenotype correlation imply a role for environmental triggers. Mechanistic studies have been limited by the absence of a cALD phenotype in the Abcd1-null mouse. Methods: We generated a cALD phenotype in 8-week-old, male Abcd1-null mice by deploying a two-hit method that combines cuprizone (CPZ) and experimental autoimmune encephalomyelitis (EAE) models. We employed in vivo MRI and post-mortem immunohistochemistry to evaluate myelin loss, astrogliosis, blood-brain barrier (BBB) disruption, immune cell infiltration, fibrin deposition, oxidative stress, and Nlrp3 inflammasome activation in mice. We used bead-based immunoassay and immunohistochemistry to evaluate IL-18 in CSF and post-mortem human cALD brain tissue. Results: MRI studies revealed T2 hyperintensities and post-gadolinium enhancement in the medial corpus callosum of cALD mice, similar to human cALD lesions. Both human and mouse cALD lesions shared common histologic features of myelin phagocytosis, myelin loss, abundant microglial activation, T and B-cell infiltration, and astrogliosis. Compared to wild-type controls, Abcd1-null mice had more severe cerebral inflammation, demyelination, fibrin deposition, oxidative stress, and IL-18 activation. IL-18 immunoreactivity co-localized with macrophages/microglia in the perivascular region of both human and mouse brain tissue. Interpretation: This novel mouse model of cALD suggests loss of Abcd1 function predisposes to more severe cerebral inflammation, oxidative stress, fibrin deposition, and Nlrp3 pathway activation, which parallels the findings seen in humans with cALD. We expect this model to enable long-sought investigations into cALD mechanisms and accelerate development of candidate therapies for lesion prevention, cessation, and remyelination.

Oral mucosal breaks trigger anti-citrullinated bacterial and human protein antibody responses in rheumatoid arthritis.

Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15+HLADRhi and CD48highS100A2pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens.

Cytotoxic CD8+ T cells target citrullinated antigens in rheumatoid arthritis.

The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8+ T cells have been described in RA, their function in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8+ T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB+CD8+ subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK+CD8+ memory subpopulation comprises smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB+CD8+ T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB+CD8+ cells are present in RA synovium. These findings suggest that cytotoxic CD8+ T cells targeting citrullinated antigens contribute to synovitis and joint tissue destruction in ACPA+ RA.

RNA-seq characterization of histamine-releasing mast cells as potential therapeutic target of osteoarthritis.

OBJECTIVE: Mast cells in the osteoarthritis (OA) synovium correlate with disease severity. This study aimed to further elucidate the role of mast cells in OA by RNA-Seq analysis and pharmacological blockade of the activity of histamine, a key mast cell mediator, in murine OA. METHODS: We examined OA synovial tissues and fluids by flow cytometry, immunostaining, single-cell and bulk RNA-Seq, qPCR, and ELISA. Cetirizine, a histamine H1 receptor (H1R) antagonist, was used to treat the destabilization of the medial meniscus (DMM) mouse model of OA. RESULTS: Flow cytometry and immunohistology analysis of OA synovial cells revealed KIT+ FcεRI+ and TPSAB1+ mast cells. Single-cell RNA-Seq of OA synovial cells identified the expression of prototypical mast cell markers KIT, TPSAB1, CPA3 and HDC, as well as distinctive markers HPGD, CAVIN2, IL1RL1, PRG2, and CKLF, confirmed by bulk RNA-Seq and qPCR. A mast cell prototypical marker expression score classified 40 OA patients into three synovial pathotypes: mast cell-high, -medium, and -low. Additionally, we detected mast cell mediators including histamine, tryptase AB1, CPA3, PRG2, CAVIN2, and CKLF in OA synovial fluids. Elevated H1R expression was detected in human OA synovium, and treatment of mice with the H1 receptor antagonist cetirizine reduced the severity and OA-related mediators in DMM. CONCLUSION: Based on differential expression of prototypical and distinct mast cell markers, human OA joints can be stratified into mast cell-high, -medium, and -low synovial tissue pathotypes. Pharmacologic blockade of histamine activity holds the potential to improve OA disease outcome.

Soaking of Autologous Tendon Grafts in Vancomycin Before Implantation Does Not Lead to Tenocyte Cytotoxicity.

BACKGROUND: Surgical site infections (SSIs) after anterior cruciate ligament (ACL) reconstruction procedures are an unfortunate complication. Soaking grafts in vancomycin before implantation has been reported to reduce the incidence of postoperative SSI after ACL reconstruction. There is potential for vancomycin to compromise graft integrity because of tenocyte toxicity. PURPOSE: To examine the in vitro toxicity of varying doses of vancomycin on human tenocytes. STUDY DESIGN: Controlled laboratory study. METHODS: Human patellar tenocytes were isolated and expanded in vitro. Tenocytes in culture were exposed to vancomycin at 5 different concentrations (400, 1600, 3200, 6400, and 12,800 µg/mL) and 3 time intervals (2, 6, and 24 hours). The control for all series was tenocyte exposure to only culture medium for each time interval. After treatment, a 10% Cell Counting Kit-8 solution in cellular growth medium was applied to the cells to examine cytotoxicity. A live/dead assay was used to assess tenocyte viability through fluorescence microscopy and flow cytometry. Results were analyzed statistically using multivariable logistic regression models with Tukey honest significant difference post hoc tests. RESULTS: Vancomycin did not cause significant changes in tenocyte viability after 2 and 6 hours of incubation at any concentration between 0 and 12,800 µg/mL. Incubation with vancomycin for 24 hours led to a significant decrease in cell viability at higher concentrations. CONCLUSION: Tenocytes derived from human patellar tendons exposed to relatively high concentrations of vancomycin for short periods of time do not demonstrate significant cell death and toxicity. CLINICAL RELEVANCE: Exposing tendons to vancomycin for a short period of time, such as before ACL reconstruction, is not likely to cause tenocyte toxicity because of vancomycin administration.

Dysregulated integrin αVß3 and CD47 signaling promotes joint inflammation, cartilage breakdown, and progression of osteoarthritis.

Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVß3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVß3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVß3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVß3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVß3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVß3 and CD47 signaling in OA pathogenesis and suggest that activation of αVß3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVß3, CD47, and their signaling pathways as a disease-modifying therapy.

Exogenous micro-RNA and antagomir modulate osteogenic gene expression in tenocytes.

Tendinopathy is a common and disabling condition that is difficult to treat. The pathomolecular events behind tendinopathy remain uncertain. Micro-RNAs (miRNAs, miRs) are short non-coding RNAs that regulate gene expression and may play a role in tendinopathy development. Tenocytes were obtained from human patellar tendons in patients undergoing anterior cruciate ligament (ACL) reconstruction. Micro-RNA mimics and antagomirs for miR-30d, 26a, and 29a were separately transfected into tenocyte culture. Gene expression for scleraxis, collagen 1 alpha 1 (COL1A1), collagen 3 alpha 1 (COL3A1), interleukin-1-beta (IL-1ß), interleukin-6 (IL-6), bone morphogenic protein 2 (BMP2), bone morphogenic protein 12 (BMP12), and osteocalcin was determined for each miRNA mimic and antagomir transfection using real-time quantitative PCR (qPCR). The results showed that exogenous miR-29a downregulated BMP2 and BMP12, while miR-26a and miR-30d did not have a significant effect on tenocyte gene expression. These findings suggest miR-29a contributes to tendon homeostasis and can serve as a potential therapeutic target in treating tendinopathy.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

BACKGROUND: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. METHODS: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. RESULTS: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1ß, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. CONCLUSIONS: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

Nicotine drives neutrophil extracellular traps formation and accelerates collagen-induced arthritis.

Objectives: The aim was to investigate the effects of nicotine on neutrophil extracellular traps (NETs) formation in current and non-smokers and on a murine model of RA. Methods: We compared spontaneous and phorbol 12-myristate 13-acetate-induced NETosis between current and non-smokers by DNA release binding. Nicotine-induced NETosis from non-smokers was assessed by DNA release binding, NET-specific (myeloperoxidase (MPO)-DNA complex) ELISA and real-time fluorescence microscopy. We also used immunofluorescent staining to detect nicotinic acetylcholine receptors (nAChRs) on neutrophils and performed a functional analysis to assess the role of nAChRs in nicotine-induced NETosis. Finally, we investigated the effects of systemic nicotine exposure on arthritis severity and NETosis in the CIA mouse model. Results: Neutrophils derived from current smokers displayed elevated levels of spontaneous and phorbol 12-myristate 13-acetate-induced NETosis. Nicotine induced dose-dependent NETosis in ex vivo neutrophils from healthy non-smokers, and co-incubation with ACPA-immune complexes or TNF-α facilitated a synergistic effect on NETosis. Real-time fluorescence microscopy revealed robust formation of NET-like structures in nicotine-exposed neutrophils. Immunofluorescent staining demonstrated the presence of the α7 subunit of the nAChR on neutrophils. Stimulation of neutrophils with an α7-specific nAChR agonist induced NETosis, whereas pretreatment with an nAChR antagonist attenuated nicotine-induced NETosis. Nicotine administration to mice with CIA exacerbated inflammatory arthritis, with higher plasma levels of NET-associated MPO-DNA complex. Conclusion: We demonstrate that nicotine is a potent inducer of NETosis, which may play an important role in accelerating arthritis in the CIA model. This study generates awareness of and the mechanisms by which nicotine-containing products, including e-cigarettes, may have deleterious effects on patients with RA.

Absence of keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice.

Human mutations in keratin 8 (K8) and keratin 18 (K18), the intermediate filament proteins of hepatocytes, predispose to several liver diseases. K8-null mice develop chronic liver injury and fragile hepatocytes, dysfunctional mitochondria, and Th2-type colitis. We tested the hypothesis that autoantibody formation accompanies the liver damage that associates with K8/K18 absence. Sera from wild-type control, K8-null, and K18-null mice were analyzed by immunoblotting and immunofluorescence staining of cell and mouse tissue homogenates. Autoantibodies to several antigens were identified in 81% of K8-null male mice 8 mo or older. Similar autoantibodies were detected in aging K18-null male mice that had a related liver phenotype but normal colon compared with K8-null mice, suggesting that the autoantibodies are linked to liver rather than colonic disease. However, these autoantibodies were not observed in nontransgenic mice subjected to 4 chronic injury models. The autoantigens are ubiquitous and partition with mitochondria. Mass spectrometry and purified protein analysis identified, mitochondrial HMG-CoA synthase, aldehyde dehydrogenase, and catalase as the primary autoantigens, and glutamate dehydrogenase and epoxide hydrolase-2 as additional autoantigens. Therefore, absence of the hepatocyte keratins results in production of anti-mitochondrial autoantibodies (AMA) that recognize proteins involved in energy metabolism and oxidative stress, raising the possibility that AMA may be found in patients with keratin mutations that associate with liver and other diseases.

Local Joint inflammation and histone citrullination in a murine model of the transition from preclinical autoimmunity to inflammatory arthritis.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are characteristic of rheumatoid arthritis (RA). However, their presence years before the onset of clinical RA is perplexing. Although multiple putative citrullinated antigens have been identified, no studies have demonstrated the specific capacity of these antigens to initiate inflammatory arthritis. This study was undertaken to recapitulate the transition from preclinical to clinical RA and to demonstrate the capacity of local citrullination to facilitate this transition. METHODS: We performed proteomic analysis of activated human neutrophils to identify citrullinated proteins, including those targeted as part of the RA immune response. Using enzyme-linked immunosorbent assay, we compared RA and osteoarthritis synovial fluid for levels of citrullinated histone H2B and its immune complex. Using macrophage activation assays, we assessed the effect of histone citrullination on immunostimulatory capacity and evaluated the stimulatory capacity of native and citrullinated H2B immune complexes. Finally, we assessed the potential for anti-citrullinated H2B antibodies to mediate arthritis in vivo. RESULTS: We identified robust targeting of neutrophil-derived citrullinated histones by the ACPA immune response. More than 90% of the RA patients had anti-citrullinated H2B antibodies. Histone citrullination increased innate immunostimulatory capacity, and immune complexes containing citrullinated histones activated macrophage cytokine production and propagated neutrophil activation. Finally, we demonstrated that immunization with H2B was arthritogenic, but only in the setting of underlying articular inflammation. CONCLUSION: Our findings indicate that citrullinated histones, specifically citrullinated H2B, are an antigenic target of the ACPA immune response. Furthermore, local generation of citrullinated antigen during low-grade articular inflammation provides a mechanistic model for the conversion from preclinical autoimmunity to inflammatory arthritis.

Brief report: citrullination within the atherosclerotic plaque: a potential target for the anti-citrullinated protein antibody response in rheumatoid arthritis.

OBJECTIVE: To investigate whether citrullinated proteins within the atherosclerotic plaque can be targeted by anti-citrullinated protein antibodies (ACPAs), forming stimulatory immune complexes that propagate the progression of atherosclerosis. METHODS: Protein lysates prepared from atherosclerotic segments of human aorta were assessed for the presence of citrulline-modified proteins, and specifically citrullinated fibrinogen (Cit-fibrinogen), by immunoprecipitation and/or immunoblotting followed by mass spectrometry. Immunohistochemical analysis of coronary artery plaque was performed to determine the presence of citrullinated proteins and peptidylarginine deiminase type 4 (PAD-4). Serum levels of anti-cyclic citrullinated peptide (anti-CCP), anti-citrullinated vimentin (anti-Cit-vimentin), and anti-Cit-fibrinogen antibodies were measured in 134 women with seropositive rheumatoid arthritis; these subjects had previously been characterized for the presence of subclinical atherosclerosis, by electron beam computed tomography scanning. RESULTS: Western blot analysis of atherosclerotic plaque lysates demonstrated several citrullinated proteins, and the presence of Cit-fibrinogen was confirmed by immunoprecipitation and mass spectrometry. Immunohistochemical analysis showed colocalization of citrullinated proteins and PAD-4 within the coronary artery plaque. In age-adjusted regression models, antibodies targeting Cit-fibrinogen and Cit-vimentin, but not CCP-2, were associated with an increased aortic plaque burden. CONCLUSION: Citrullinated proteins are prevalent within atherosclerotic plaques, and certain ACPAs are associated with the atherosclerotic burden. These observations suggest that targeting of citrullinated epitopes, specifically Cit-fibrinogen, within atherosclerotic plaques could provide a mechanism for the accelerated atherosclerosis observed in patients with RA.

Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems.

Glycolysis is the initial step of glucose catabolism and is up-regulated in cancer cells (the Warburg Effect). Such shifts toward a glycolytic phenotype have not been explored widely in other biological systems, and the molecular mechanisms underlying the shifts remain unknown. With proteomics, we observed increased glycolysis in disused human diaphragm muscle. In disused muscle, lung cancer, and H(2)O(2)-treated myotubes, we show up-regulation of the rate-limiting glycolytic enzyme muscle-type phosphofructokinase (PFKm, >2 fold, P<0.05) and accumulation of lactate (>150%, P<0.05). Using microRNA profiling, we identify miR-320a as a regulator of PFKm expression. Reduced miR-320a levels (to ∼50% of control, P<0.05) are associated with the increased PFKm in each of these diverse systems. Manipulation of miR-320a levels both in vitro and in vivo alters PFKm and lactate levels in the expected directions. Further, miR-320a appears to regulate oxidative stress-induced PFKm expression, and reduced miR-320a allows greater induction of glycolysis in response to H(2)O(2) treatment. We show that this microRNA-mediated regulation occurs through PFKm's 3' untranslated region and that Ets proteins are involved in the regulation of PFKm via miR-320a. These findings suggest that oxidative stress-responsive microRNA-320a may regulate glycolysis broadly within nature.

Plasma proteins present in osteoarthritic synovial fluid can stimulate cytokine production via Toll-like receptor 4.

INTRODUCTION: Osteoarthritis (OA) is a degenerative disease characterized by cartilage breakdown in the synovial joints. The presence of low-grade inflammation in OA joints is receiving increasing attention, with synovitis shown to be present even in the early stages of the disease. How the synovial inflammation arises is unclear, but proteins in the synovial fluid of affected joints could conceivably contribute. We therefore surveyed the proteins present in OA synovial fluid and assessed their immunostimulatory properties. METHODS: We used mass spectrometry to survey the proteins present in the synovial fluid of patients with knee OA. We used a multiplex bead-based immunoassay to measure levels of inflammatory cytokines in serum and synovial fluid from patients with knee OA and from patients with rheumatoid arthritis (RA), as well as in sera from healthy individuals. Significant differences in cytokine levels between groups were determined by significance analysis of microarrays, and relations were determined by unsupervised hierarchic clustering. To assess the immunostimulatory properties of a subset of the identified proteins, we tested the proteins' ability to induce the production of inflammatory cytokines by macrophages. For proteins found to be stimulatory, the macrophage stimulation assays were repeated by using Toll-like receptor 4 (TLR4)-deficient macrophages. RESULTS: We identified 108 proteins in OA synovial fluid, including plasma proteins, serine protease inhibitors, proteins indicative of cartilage turnover, and proteins involved in inflammation and immunity. Multiplex cytokine analysis revealed that levels of several inflammatory cytokines were significantly higher in OA sera than in normal sera, and levels of inflammatory cytokines in synovial fluid and serum were, as expected, higher in RA samples than in OA samples. As much as 36% of the proteins identified in OA synovial fluid were plasma proteins. Testing a subset of these plasma proteins in macrophage stimulation assays, we found that Gc-globulin, α1-microglobulin, and α2-macroglobulin can signal via TLR4 to induce macrophage production of inflammatory cytokines implicated in OA. CONCLUSIONS: Our findings suggest that plasma proteins present in OA synovial fluid, whether through exudation from plasma or production by synovial tissues, could contribute to low-grade inflammation in OA by functioning as so-called damage-associated molecular patterns in the synovial joint.

Chaperone activity of α B-crystallin is responsible for its incorrect assignment as an autoantigen in multiple sclerosis.

For 15 y, α B-crystallin (heat shock protein [Hsp] B5) has been labeled an autoantigen in multiple sclerosis (MS) based on humoral and cellular responses found in humans and animal models. However, there have been several scientific inconsistencies with this assignment, ranging from studies demonstrating small differences in anticrystallin responses between patients and healthy individuals to the inability of crystallin-specific T cells to induce symptoms of experimental allergic encephalomyelitis in animal models. Experiments in this article demonstrate that the putative anti-HspB5 Abs from 23 MS patients cross-react with 7 other members of the human small Hsp family and were equally present in normal plasma. Biolayer interferometry demonstrates that the binding was temperature dependent, and that the calculated K(a) increased as the concentration of the sHsp decreased. These two patterns are characteristic of multiple binding sites with varying affinities, the composition of which changes with temperature, supporting the hypothesis that HspB5 bound the Ab and not the reverse. HspB5 also precipitated Ig heavy and L chains from sera from patients with MS. These results establish that small Hsps bind Igs with high affinity and refute much of the serological data used to assign α B-crystallin as an autoantigen.

c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis.

INTRODUCTION: Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis. METHODS: We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA. RESULTS: GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid. CONCLUSIONS: These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.

Autoimmunity against fibrinogen mediates inflammatory arthritis in mice.

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the presence of anticitrullinated protein Abs, although the exact targets and role of anticitrullinated protein autoimmunity in the pathogenesis of RA remain to be defined. Fibrinogen, which can be citrullinated, has recently emerged as a candidate autoantigen. To determine whether autoimmunity against fibrinogen can mediate inflammatory arthritis, we immunized a variety of common mouse strains with fibrinogen and found that DBA/1 and SJL mice developed an inflammatory and erosive arthritis. Mice with fibrinogen-induced arthritis (FIA) possess fibrinogen-reactive T cells that produce the proinflammatory cytokines IL-6, IL-17, TNF-alpha, and IFN-gamma. FIA can be adoptively transferred with either plasma or fibrinogen-specific T cells from diseased mice. Mice with FIA possess rheumatoid factor, circulating immune complexes, and anticyclic citrullinated peptide Abs, all of which are characteristic of human RA. These observations demonstrate that fibrinogen is arthritogenic in mice and that the pathogenesis of FIA is mediated by both autoantibodies and fibrinogen-reactive T cells.

Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin.

CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of B-lymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin-binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. After engagement of CD81, it colocalized with ezrin and F-actin, and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This mechanism might explain the pleiotropic effects induced in response to stimulation of cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU-rich elements.

The 3'untranslated region (UTR) of human LDL receptor (LDLR) mRNA contains three AU-rich elements (AREs) responsible for rapid mRNA turnover and mediates the stabilization induced by berberine (BBR). However, the identities of the specific RNA binding proteins involved in the regulation of LDLR mRNA stability at the steady state level or upon BBR treatment are unknown. By conducting small interfering RNA library screenings, biotinylated RNA pull-down, mass spectrometry analysis, and functional assays, we now identify heterogeneous nuclear ribonucleoprotein D (hnRNP D), hnRNP I, and KH-type splicing regulatory protein (KSRP) as key modulators of LDLR mRNA stability in liver cells. We show that hnRNP D, I, and KSRP interact with AREs of the LDLR 3'UTR with sequence specificity. Silencing the expression of these proteins increased LDLR mRNA and protein levels. We further demonstrate that BBR-induced mRNA stabilization involves hnRNP I and KSRP, as their cellular depletions abolished the BBR effect and BBR treatment reduced the binding of hnRNP I and KSRP to the LDLR mRNA 3'UTR. These new findings demonstrate that LDLR mRNA stability is controlled by a group of ARE binding proteins, including hnRNP D, hnRNP I, and KSRP. Our results suggest that interference with the ability of destabilizing ARE binding proteins to interact with LDLR-ARE motifs is likely a mechanism for regulating LDLR expression by compounds such as BBR and perhaps others.

Epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination.

INTRODUCTION: Anti-citrullinated protein antibodies have a diagnostic role in rheumatoid arthritis (RA); however, little is known about their origins and contribution to pathogenesis. Citrullination is the post-translational conversion of arginine to citrulline by peptidyl arginine deiminase, and increased citrullination of proteins is observed in the joint tissue in RA and in brain tissue in multiple sclerosis (MS). METHODS: We applied synovial and myelin protein arrays to examine epitope spreading of B cell responses to citrullinated epitopes in both the collagen-induced arthritis (CIA) model for RA and the experimental autoimmune encephalomyelitis (EAE) model for MS. Synovial and myelin protein arrays contain a spectrum of proteins and peptides, including native and citrullinated forms, representing candidate autoantigens in RA and MS, respectively. We applied these arrays to characterise the specificity of autoantibodies in serial serum samples derived from mice with acute and chronic stages of CIA and EAE. RESULTS: In samples from pre-disease CIA and acute-disease EAE, we observed autoantibody targeting of the immunising antigen and responses to a limited set of citrullinated epitopes. Over the course of diseases, the autoantibody responses expanded to target multiple citrullinated epitopes in both CIA and EAE. Using immunoblotting and mass spectrometry analysis, we identified citrullination of multiple polypeptides in CIA joint and EAE brain tissue that have not previously been described as citrullinated. CONCLUSIONS: Our results suggest that anti-citrulline antibody responses develop in the early stages of CIA and EAE, and that autoimmune inflammation results in citrullination of joint proteins in CIA and brain proteins in EAE, thereby creating neoantigens that become additional targets in epitope spreading of autoimmune responses.