The cholinergic anti-inflammatory pathway inhibits inflammation without lymphocyte relay.

The magnitude of innate inflammatory immune responses is dependent on interactions between peripheral neural and immune cells. In particular, a cholinergic anti-inflammatory pathway (CAP) has been identified in the spleen whereby noradrenaline (NA) released by splenic nerves binds to ß2-adrenergic receptors (ß2-AR) on CD4+ T cells which, in turn, release acetylcholine (ACh). The binding of ACh to α7 acetylcholine receptors (α7-AChR) expressed by splenic macrophages inhibits the production of inflammatory cytokines, including tumor necrosis factor (TNF). However, the role of ACh-secreting CD4+ T-cells in the CAP is still controversial and largely based on the absence of this anti-inflammatory pathway in mice lacking T-cells (nude, FoxN1-/-). Using four conscious, non-lymphopenic transgenic mouse models, we found that, rather than acting on CD4+ T-cells, NA released by splenic nerve terminals acts directly onto ß2-AR on splenic myeloid cells to exert this anti-inflammatory effect. We also show that, while larger doses of LPS are needed to trigger CAP in nude mouse strain compared to other strains, TNF production can be inhibited in these animals lacking CD4+ T-cell by stimulating either the vagus or the splenic nerve. We demonstrate that CD4+ T-cells are dispensable for the CAP after antibody-mediated CD4+ T-cell depletion in wild type mice. Furthermore, we found that NA-mediated inhibition of in vitro LPS-induced TNF secretion by human or porcine splenocytes does not require α7-AChR signaling. Altogether our data demonstrate that activation of the CAP by stimulation of vagus or splenic nerves in mice is mainly mediated by direct binding of NA to ß2-AR on splenic macrophages, and suggest that the same mechanism is at play in larger species.

Electrical stimulation of the splenic nerve bundle ameliorates dextran sulfate sodium-induced colitis in mice.

BACKGROUND: Vagus nerve stimulation has been suggested to affect immune responses, partly through a neuronal circuit requiring sympathetic innervation of the splenic nerve bundle and norepinephrine (NE) release. Molecular and cellular mechanisms of action remain elusive. Here, we investigated the therapeutic value of this neuromodulation in inflammatory bowel disease (IBD) by applying electrical splenic nerve bundle stimulation (SpNS) in mice with dextran sulfate sodium (DSS)-induced colitis. METHODS: Cuff electrodes were implanted around the splenic nerve bundle in mice, whereupon mice received SpNS or sham stimulation. Stimulation was applied 6 times daily for 12 days during DSS-induced colitis. Colonic and splenic tissues were collected for transcriptional analyses by qPCR and RNA-sequencing (RNA-seq). In addition, murine and human splenocytes were stimulated with lipopolysaccharide (LPS) in the absence or presence of NE. Single-cell RNA-seq data from publicly available data sets were analyzed for expression of ß-adrenergic receptors (ß-ARs). RESULTS: Colitic mice undergoing SpNS displayed reduced colon weight/length ratios and showed improved Disease Activity Index scores with reduced Tumor Necrosis Factor α mRNA expression in the colon compared with sham stimulated mice. Analyses of splenocytes from SpNS mice using RNA-seq demonstrated specific immune metabolism transcriptome profile changes in myeloid cells. Splenocytes showed expression of ß-ARs in myeloid and T cells. Cytokine production was reduced by NE in mouse and human LPS-stimulated splenocytes. CONCLUSIONS: Together, our results demonstrate that SpNS reduces clinical features of colonic inflammation in mice with DSS-induced colitis possibly by inhibiting splenic myeloid cell activation. Our data further support exploration of the clinical use of SpNS for patients with IBD.

Splenic arterial neurovascular bundle stimulation in esophagectomy: A feasibility and safety prospective cohort study.

Introduction: The autonomic nervous system is a key regulator of inflammation. Electrical stimulation of the vagus nerve has been shown to have some preclinical efficacy. However, only a few clinical studies have been reported to treat inflammatory diseases. The present study evaluates, for the first time, neuromodulation of the splenic arterial neurovascular bundle (SpA NVB) in patients undergoing minimally invasive esophagectomy (MIE), in which the SpA NVB is exposed as part of the procedure. Methods: This single-center, single-arm study enrolled 13 patients undergoing MIE. During the abdominal phase of the MIE, a novel cuff was placed around the SpA NVB, and stimulation was applied. The primary endpoint was the feasibility and safety of cuff application and removal. A secondary endpoint included the impact of stimulation on SpA blood flow changes during the stimulation, and an exploratory point was C-reactive protein (CRP) levels on postoperative day (POD) 2 and 3. Results: All patients successfully underwent placement, stimulation, and removal of the cuff on the SpA NVB with no adverse events related to the investigational procedure. Stimulation was associated with an overall reduction in splenic arterial blood flow but not with changes in blood pressure or heart rate. When compared to historic Propensity Score Matched (PSM) controls, CRP levels on POD2 (124 vs. 197 mg/ml, p = 0.032) and POD3 (151 vs. 221 mg/ml, p = 0.033) were lower in patients receiving stimulation. Conclusion: This first-in-human study demonstrated for the first time that applying a cuff around the SpA NVB and subsequent stimulation is safe, feasible, and may have an effect on the postoperative inflammatory response following MIE. These findings suggest that SpA NVB stimulation may offer a new method for immunomodulatory therapy in acute or chronic inflammatory conditions.

'SMASH' recommendations for standardised microscopic arthritis scoring of histological sections from inflammatory arthritis animal models.

Animal models for inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis are widely accepted and frequently used to identify pathological mechanisms and validate novel therapeutic strategies. Unfortunately, many publications reporting on these animal studies lack detailed description and appropriate assessment of the distinct histopathological features of arthritis: joint inflammation, cartilage damage and bone erosion. Therefore, the European consortium BeTheCure, consisting of 38 academic and industrial partners from 15 countries, set as goal to standardise the histological evaluation of joint sections from animal models of inflammatory arthritis. The consensual approach of a task force including 16 academic and industrial scientists as well as laboratory technicians has resulted in the development of the Standardised Microscopic Arthritis Scoring of Histological sections ('SMASH') recommendations for a standardised processing and microscopic scoring of the characteristic histopathological features of arthritis, exemplified by four different rodent models for arthritis: murine collagen-induced arthritis, collagen-antibody-induced arthritis, human tumour necrosis factor transgenic Tg197 mice and rat pristane-induced arthritis, applicable to any other inflammatory arthritis model. Through standardisation, the SMASH recommendations are designed to improve and maximise the information derived from in vivo arthritis experiments and to promote reproducibility and transparent reporting on such studies. In this manuscript, we will discuss and provide recommendations for analysis of histological joint sections: identification of the regions of interest, sample preparation, staining procedures and quantitative scoring methods. In conclusion, awareness of the different features of the arthritis pathology in animal models of inflammatory arthritis is of utmost importance for reliable research outcome, and the standardised histological processing and scoring methods in these SMASH recommendations will help increase uniformity and reproducibility in preclinical research on inflammatory arthritis.

Morphometric analysis of the splenic artery using contrast-enhanced computed tomography (CT).

PURPOSE: To evaluate the morphology and course of the splenic artery, which might impact the surgical implantation of systems that stimulate the nerves surrounding the splenic artery. Experimental studies indicate that these nerves play an important part in immune modulation, and might be a potential target in the treatment of autoimmune diseases. METHODS: This retrospective cohort study made use of contrast-enhanced CT images from 40 male and 40 female patients (age 30-69) that underwent a CT examination of the aorta, kidneys or pancreas. Anatomic features were described including total splenic artery length, calibers, tortuosity, the presence of arterial loops and the branching pattern of the splenic artery. RESULTS: No age-gender-related differences could be found related to tortuosity or branching pattern. The length of splenic artery in contact with pancreatic tissue decreased with increasing age, but was not different between genders. Artery diameters were wider in male compared to female subjects. Loops of variable directions, that represent a part of the artery that curls out of the pancreatic tissue, were identified in each age-gender category and were present in nearly all subjects (86%). CONCLUSION: This study suggests that although some anatomic features of the splenic artery are subject to factors as age and gender, the tortuosity of the splenic artery is not age dependent. Most subjects had one or multiple loops, which can serve as a target for neuromodulatory devices. Future studies should investigate whether splenic nerve stimulation is safe and feasible.

Neuroimmune Interactions in the Gut and Their Significance for Intestinal Immunity.

Inflammatory bowel diseases (IBD) have a complex, multifactorial pathophysiology with an unmet need for effective treatment. This calls for novel strategies to improve disease outcome and quality of life for patients. Increasing evidence suggests that autonomic nerves and neurotransmitters, as well as neuropeptides, modulate the intestinal immune system, and thereby regulate the intestinal inflammatory processes. Although the autonomic nervous system is classically divided in a sympathetic and parasympathetic branch, both play a pivotal role in the crosstalk with the immune system, with the enteric nervous system acting as a potential interface. Pilot clinical trials that employ vagus nerve stimulation to reduce inflammation are met with promising results. In this paper, we review current knowledge on the innervation of the gut, the potential of cholinergic and adrenergic systems to modulate intestinal immunity, and comment on ongoing developments in clinical trials.

Apical splenic nerve electrical stimulation discloses an anti-inflammatory pathway relying on adrenergic and nicotinic receptors in myeloid cells.

The autonomic nervous system innervates all lymphoid tissues including the spleen therefore providing a link between the central nervous system and the immune system. The only known mechanism of neural inhibition of inflammation in the spleen relies on the production of norepinephrine by splenic catecholaminergic fibers which binds to ß2-adrenergic receptors (ß 2-ARs) of CD4+ T cells. These CD4+ T cells trigger the release of acetylcholine that inhibits the secretion of inflammatory cytokines by macrophages through α7 nicotinic acetylcholine receptor (α7nAchRs) signaling. While the vagal anti-inflammatory pathway has been extensively studied in rodents, it remains to be determined whether it coexists with other neural pathways. Here, we have found that three nerve branches project to the spleen in mice. While two of these nerves are associated with an artery and contain catecholaminergic fibers, the third is located at the apex of the spleen and contain both catecholaminergic and cholinergic fibers. We found that electrical stimulation of the apical nerve, but not the arterial nerves, inhibited inflammation independently of lymphocytes. In striking contrast to the anti-inflammatory pathway mechanism described so far, we also found that the inhibition of inflammation by apical nerve electrical stimulation relied on signaling by both ß 2-ARs and α7nAchRs in myeloid cells, with these two signaling pathways acting in parallel. Most importantly, apical splenic nerve electrical stimulation mitigated clinical symptoms in a mouse model of rheumatoid arthritis further providing the proof-of-concept that such an approach could be beneficial in patients with Immune-mediated inflammatory diseases.

Loss of intestinal sympathetic innervation elicits an innate immune driven colitis.

BACKGROUND: Both the parasympathetic and sympathetic nervous system exert control over innate immune responses. In inflammatory bowel disease, sympathetic innervation in intestinal mucosa is reduced. Our aim was to investigate the role of sympathetic innervation to the intestine on regulation of the innate immune responses. METHODS: In lipopolysaccharide (LPS)-stimulated macrophages, we evaluated the effect of adrenergic receptor activation on cytokine production and metabolic profile. In vivo, the effect of sympathetic denervation on mucosal innate immune responses using 6-hydroxydopamine (6-OHDA), or using surgical transection of the superior mesenteric nerve (sympathectomy) was tested in Rag1-/- mice that lack T- and B-lymphocytes. RESULTS: In murine macrophages, adrenergic ß2 receptor activation elicited a dose-dependent reduction of LPS-induced cytokines, reduced LPS-induced glycolysis and increased maximum respiration. Sympathectomy led to a significantly decreased norepinephrine concentration in intestinal tissue. Within 14 days after sympathectomy, mice developed clinical signs of colitis, colon oedema and excess colonic cytokine production. Both 6-OHDA and sympathectomy led to prominent goblet cell depletion and histological damage of colonic mucosa. CONCLUSIONS: We conclude that the sympathetic nervous system plays a regulatory role in constraining innate immune cell reactivity towards microbial challenges, likely via the adrenergic ß2 receptor.

Empty Capsids and Macrophage Inhibition/Depletion Increase rAAV Transgene Expression in Joints of Both Healthy and Arthritic Mice.

Gene therapy has potential to treat rheumatic diseases; however, the presence of macrophages in the joint might hamper adeno-associated viral vector-mediated gene delivery. Here we demonstrate that in arthritic, but also in healthy, mice administration of agents that influence macrophage activity/number and/or addition of empty decoy capsids substantially improve the efficacy of recombinant adeno-associated viral vector 5 transgene expression in the joint. Pretreatment with triamcinolone or clodronate liposomes improved luciferase expression over a period of 4 weeks. Similar results were seen when empty decoy capsids were added to full genome containing capsids in a 5:1 ratio. In a study to assess the duration of expression as well as to investigate the combination of these two approaches, we observed a synergistic enhancement of gene expression, sustained for at least 12 weeks. The enhancement of gene expression was independent of the route of administration of triamcinolone (intra-articular or intramuscular). In healthy mice it was demonstrated that the combination improved expression of the transgene significantly, in a serotype independent manner. These data have implications for future applications of gene therapy to the joint and for other tissues with an abundance of macrophages.

Single-joint Assessment for the Evaluation of Intraarticular Treatment: Responsiveness and Discrimination of the Composite Change Index.

OBJECTIVE: To investigate responsiveness, discrimination, and construct validity of a composite change index (CCI) for the assessment of single-joint involvement in inflammatory arthritis. METHODS: Evaluation of standardized response means (SRM), Guyatt effect size, and Spearman rank correlation coefficient in a randomized controlled trial investigating the effect of an intraarticular etanercept injection. RESULTS: The CCI showed a high SRM (1.68) and high Guyatt effect size (2.72). Both visual analog scale of pain and functionality had a moderate Guyatt effect size (2.06, 2.44) and high SRM (0.81, 0.97). CONCLUSION: This study supports the use of the CCI as a single-joint assessment after single-joint intervention. CLINICAL TRIAL REGISTRATION: NTR-1210.

Preclinical Potency and Biodistribution Studies of an AAV 5 Vector Expressing Human Interferon-ß (ART-I02) for Local Treatment of Patients with Rheumatoid Arthritis.

INTRODUCTION: Proof of concept for local gene therapy for the treatment of arthritis with immunomodulatory cytokine interferon beta (IFN-ß) has shown promising results in animal models of rheumatoid arthritis (RA). For the treatment of RA patients, we engineered a recombinant adeno-associated serotype 5 vector (rAAV5) encoding human (h)IFN-ß under control of a nuclear factor κB promoter (ART-I02). METHODS: The potency of ART-I02 in vitro as well as biodistribution in vivo in arthritic animals was evaluated to characterize the vector prior to clinical application. ART-I02 expression and bioactivity after transduction was evaluated in fibroblast-like synoviocytes (FLS) from different species. Biodistribution of the vector after local injection was assessed in a rat adjuvant arthritis model through qPCR analysis of vector DNA. In vivo imaging was used to investigate transgene expression and kinetics in a mouse collagen induced arthritis model. RESULTS: Transduction of RA FLS in vitro with ART-I02 resulted in high expression levels of bioactive hIFN-ß. Transduction of FLS from rhesus monkeys, rodents and rabbits with ART-I02 showed high transgene expression, and hIFN-ß proved bioactive in FLS from rhesus monkeys. Transgene expression and bioactivity in RA FLS were unaltered in the presence of methotrexate. In vivo, vector biodistribution analysis in rats after intra-articular injection of ART-I02 demonstrated that the majority of vector DNA remained in the joint (>93%). In vivo imaging in mice confirmed local expression of rAAV5 in the knee joint region and demonstrated rapid detectable and sustained expression up until 7 weeks. CONCLUSIONS: These data show that hIFN-ß produced by RA FLS transduced with ART-I02 is bioactive and that intra-articular delivery of rAAV5 drives expression of a therapeutic transgene in the joint, with only limited biodistribution of vector DNA to other tissues, supporting progress towards a phase 1 clinical trial for the local treatment of arthritis in patients with RA.

Safety, Biodistribution, and Efficacy of an AAV-5 Vector Encoding Human Interferon-Beta (ART-I02) Delivered via Intra-Articular Injection in Rhesus Monkeys with Collagen-Induced Arthritis.

Preclinical studies to assess biodistribution, safety, and initial efficacy of ART-I02, an adeno-associated type 5 (rAAV5) vector expressing human interferon ß (hIFN-ß), were performed in a total of 24 rhesus monkeys with collagen-induced arthritis. All monkeys were naïve or showed limited neutralizing antibody (Nab) titers to AAV5 at the start of the study. Animals were injected with a single intra-articular dose of ART-I02 or placebo, consisting of 3.2×10(13) vg (Dose A=maximum feasible dose), 4.58×10(12) vg (Dose B), or placebo in the first affected finger joint, the ipsilateral knee, and ankle joint at the same time point. Animals were monitored for clinical parameters and well-being with a maximum of 4 weeks, with the option that the severity of arthritis could necessitate an earlier time point of sacrifice. No adverse events were noted after injection of ART-I02. No abnormalities were observed after histological evaluation of all organs. At both dose levels, immunohistochemical staining indicated expression of hIFN-ß. In animals injected with Dose A, we observed stabilization or a reduction in swelling in the finger joint in which vector was administered. The highest copy numbers of vector DNA were detected in synovial tissue of the injected joint and the draining lymph node of the injected knee. High titers of Nab to rAAV5 were observed at the end of the study. Five monkeys developed an rAAV5-specific T-cell response. Two monkeys developed Nab to hIFN-ß. In conclusion, intra-articular injection of ART-I02 was well-tolerated and did not induce adverse events. After administration of Dose A of ART-I02, we observed a beneficial effect on joint swelling, substantiated by decreased histological inflammation and bone erosion scores. A GMP vector for clinical application has been manufactured and is currently being tested in GLP rodent studies, with the aim to move forward to a clinical trial.

Two novel α7 nicotinic acetylcholine receptor ligands: in vitro properties and their efficacy in collagen-induced arthritis in mice.

INTRODUCTION: The cholinergic anti-inflammatory pathway can downregulate inflammation via the release of acetylcholine (ACh) by the vagus nerve. This neurotransmitter binds to the α7 subunit of nicotinic acetylcholine receptors (α7nAChR), expressed on macrophages and other immune cells. We tested the pharmacological and functional profile of two novel compounds, PMP-311 and PMP-072 and investigated their role in modulating collagen-induced arthritis (CIA) in mice. METHODS: Both compounds were characterized with binding, electrophysiological, and pharmacokinetic studies. For in vivo efficacy studies in the CIA model the compounds were administered daily by oral gavage from day 20 till sacrifice at day 34. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. RESULTS: Treatment with PMP-311 was effective in preventing disease onset, reducing clinical signs of arthritis, and reducing synovial inflammation and bone destruction. PMP-072 also showed a trend in arthritis reduction at all concentrations tested. The data showed that while both compounds bind to α7nAChR with high affinity, PMP-311 acts like a classical agonist of ion channel activity, and PMP-072 can actually act as an ion channel antagonist. Moreover, PMP-072 was clearly distinct from typical competitive antagonists, since it was able to act as a silent agonist. It synergizes with the allosteric modulator PNU-120596, and subsequently activates desensitized α7nAChR. However, PMP-072 was less efficacious than PMP-311 at both channel activation and desensitization, suggesting that both conducting and non-conducting states maybe of importance in driving an anti-inflammatory response. Finally, we found that the anti-arthritic effect can be observed despite limited penetration of the central nervous system. CONCLUSIONS: These data provide direct evidence that the α7nAChR in immune cells does not require typical ion channel activation to exert its antiinflammatory effects.

Neurostimulation of the cholinergic anti-inflammatory pathway ameliorates disease in rat collagen-induced arthritis.

INTRODUCTION: The inflammatory reflex is a physiological mechanism through which the nervous system maintains immunologic homeostasis by modulating innate and adaptive immunity. We postulated that the reflex might be harnessed therapeutically to reduce pathological levels of inflammation in rheumatoid arthritis by activating its prototypical efferent arm, termed the cholinergic anti-inflammatory pathway. To explore this, we determined whether electrical neurostimulation of the cholinergic anti-inflammatory pathway reduced disease severity in the collagen-induced arthritis model. METHODS: Rats implanted with vagus nerve cuff electrodes had collagen-induced arthritis induced and were followed for 15 days. Animals underwent active or sham electrical stimulation once daily from day 9 through the conclusion of the study. Joint swelling, histology, and levels of cytokines and bone metabolism mediators were assessed. RESULTS: Compared with sham treatment, active neurostimulation of the cholinergic anti-inflammatory pathway resulted in a 52% reduction in ankle diameter (p = 0.02), a 57% reduction in ankle diameter (area under curve; p = 0.02) and 46% reduction overall histological arthritis score (p = 0.01) with significant improvements in inflammation, pannus formation, cartilage destruction, and bone erosion (p = 0.02), accompanied by numerical reductions in systemic cytokine levels, not reaching statistical significance. Bone erosion improvement was associated with a decrease in serum levels of receptor activator of NF-κB ligand (RANKL) from 132±13 to 6±2 pg/mL (mean±SEM, p = 0.01). CONCLUSIONS: The severity of collagen-induced arthritis is reduced by neurostimulation of the cholinergic anti-inflammatory pathway delivered using an implanted electrical vagus nerve stimulation cuff electrode, and supports the rationale for testing this approach in human inflammatory disorders.

Divergent effects of endogenous and exogenous glucocorticoid-induced leucine zipper in animal models of inflammation and arthritis.

OBJECTIVE: Glucocorticoid-induced leucine zipper (GILZ) has effects on inflammatory pathways that suggest it to be a key inhibitory regulator of the immune system, and its expression is exquisitely sensitive to induction by glucocorticoids. We undertook this study to test our hypothesis that GILZ deficiency would exacerbate experimental immune-mediated inflammation and impair the effects of glucocorticoids on inflammation and, correspondingly, that exogenous GILZ would inhibit these events. METHODS: GILZ(-/-) mice were generated using the Cre/loxP system, and responses were studied in delayed-type hypersensitivity (DTH), antigen-induced arthritis (AIA), K/BxN serum-transfer arthritis, and lipopolysaccharide (LPS)-induced cytokinemia. Therapeutic expression of GILZ via administration of recombinant adeno-associated virus expressing the GILZ gene (GILZ-rAAV) was compared to the effects of glucocorticoid in collagen-induced arthritis (CIA). RESULTS: Increased T cell proliferation and DTH were observed in GILZ(-/-) mice, but neither AIA nor K/BxN serum-transfer arthritis was affected, and GILZ deficiency did not affect LPS-induced cytokinemia. Deletion of GILZ did not impair the effects of exogenous glucocorticoids on CIA or cytokinemia. In contrast, overexpression of GILZ in joints significantly inhibited CIA, with an effect similar to that of dexamethasone. CONCLUSION: Despite effects on T cell activation, GILZ deficiency had no effect on effector pathways of arthritis and was unexpectedly redundant with effects of glucocorticoids. These findings do not support the hypothesis that GILZ is central to the actions of glucocorticoids, but the efficacy of exogenous GILZ in CIA suggests that further evaluation of GILZ in inflammatory disease is required.

SNAPIN: an endogenous Toll-like receptor ligand in rheumatoid arthritis.

OBJECTIVE: The mechanisms contributing to the persistent activation of macrophages in rheumatoid arthritis (RA) are not fully understood. Some studies suggest that endogenous toll-like receptor (TLR) ligands promote the chronic inflammation observed in RA. The objective of this study was to identify endogenous TLR ligands expressed in RA synovial tissue (ST) based on their ability to bind the extracellular domains of TLR2 or TLR4. METHODS: A yeast two-hybrid cDNA library was constructed from ST obtained by arthroscopy from patients with RA and screened using the extracellular domains of TLR2 and TLR4 as the bait. Interactions between TLRs and Snapin were demonstrated by reciprocal co-immunoprecipitation. ST was examined by histology, and single- and two-colour immunohistochemistry and quantitative reverse transcriptase PCR. Snapin (SNAP - associated protein) expression in macrophages was examined by Western Blot analysis and confocal microscopy. The ability of Snapin to activate through TLR2 was examined. RESULTS: Employing a yeast two-hybrid system, Snapin was the most frequently identified molecule that interacted with TLR2. These results were confirmed by pull-down of in vitro-expressed Snapin together with TLR2. By immunohistochemistry and quantitative reverse transcriptase PCR, Snapin was highly expressed in RA ST, and it was readily detected in macrophages, where it co-localised in the late endosomes. ST Snapin expression correlated with inflammation and was not disease specific. Finally, Snapin was capable of activating through TLR2. CONCLUSION: These observations identify Snapin as a novel endogenous TLR2 ligand in RA, and thus support a role for persistent TLR2 signalling in the pathogenesis of RA.

Advancements in adeno-associated viral gene therapy approaches: exploring a new horizon.

Gene therapy is a promising new therapeutic strategy that has been explored in a wide variety of diseases, ranging from cancer to hemophilia, and ocular disorders to autoimmune diseases, among others. Proof of concept of gene transfer approaches has been shown in over 100 studies of animal models of disease, although only a few are under development for clinical application. The US Food and Drug Administration and the European Medicines Agency have not approved any viral human gene therapy products for sale so far, but the amount of gene-related research and development occurring in the United States and Europe continues to grow at a fast rate. This review summarizes the current status of developments in the field of viral gene therapy using adeno-associated virus as a vector, with a special focus on arthritis. For rheumatoid arthritis, and to a lesser extent for other immune-related inflammatory disorders, several cell and gene transfer approaches have been investigated at the preclinical level and a few have been implemented in clinical trials. Finally, both the potential and the hurdles that are faced during development of a viral gene therapy through to its clinical application are discussed.

Animal models for arthritis: innovative tools for prevention and treatment.

The development of novel treatments for rheumatoid arthritis (RA) requires the interplay between clinical observations and studies in animal models. Given the complex molecular pathogenesis and highly heterogeneous clinical picture of RA, there is an urgent need to dissect its multifactorial nature and to propose new strategies for preventive, early and curative treatments. Research on animal models has generated new knowledge on RA pathophysiology and aetiology and has provided highly successful paradigms for innovative drug development. Recent focus has shifted towards the discovery of novel biomarkers, with emphasis on presymptomatic and emerging stages of human RA, and towards addressing the pathophysiological mechanisms and subsequent efficacy of interventions that underlie different disease variants. Shifts in the current paradigms underlying RA pathogenesis have also led to increased demand for new (including humanised) animal models. There is therefore an urgent need to integrate the knowledge on human and animal models with the ultimate goal of creating a comprehensive 'pathogenesis map' that will guide alignment of existing and new animal models to the subset of disease they mimic. This requires full and standardised characterisation of all models at the genotypic, phenotypic and biomarker level, exploiting recent technological developments in 'omics' profiling and computational biology as well as state of the art bioimaging. Efficient integration and dissemination of information and resources as well as outreach to the public will be necessary to manage the plethora of data accumulated and to increase community awareness and support for innovative animal model research in rheumatology.

Restoring the balance of the autonomic nervous system as an innovative approach to the treatment of rheumatoid arthritis.

The immunomodulatory effect of the autonomic nervous system has raised considerable interest over the last decades. Studying the influence on the immune system and the role in inflammation of the sympathetic as well as the parasympathetic nervous system not only will increase our understanding of the mechanism of disease, but also could lead to the identification of potential new therapeutic targets for chronic immune-mediated inflammatory diseases, such as rheumatoid arthritis (RA). An imbalanced autonomic nervous system, with a reduced parasympathetic and increased sympathetic tone, has been a consistent finding in RA patients. Studies in animal models of arthritis have shown that influencing the sympathetic (via α- and ß-adrenergic receptors) and the parasympathetic (via the nicotinic acetylcholine receptor α7nAChR or by electrically stimulating the vagus nerve) nervous system can have a beneficial effect on inflammation markers and arthritis. The immunosuppressive effect of the parasympathetic nervous system appears less ambiguous than the immunomodulatory effect of the sympathetic nervous system, where activation can lead to increased or decreased inflammation depending on timing, doses and kind of adrenergic agent used. In this review we will discuss the current knowledge of the role of both the sympathetic (SNS) and parasympathetic nervous system (PNS) in inflammation with a special focus on the role in RA. In addition, potential antirheumatic strategies that could be developed by targeting these autonomic pathways are discussed.

Silencing the expression of Ras family GTPase homologues decreases inflammation and joint destruction in experimental arthritis.

Changes in the expression and activation status of Ras proteins are thought to contribute to the pathological phenotype of stromal fibroblast-like synoviocytes (FLS) in rheumatoid arthritis, a prototypical immune-mediated inflammatory disease. Broad inhibition of Ras and related proteins has shown protective effects in animal models of arthritis, but each of the Ras family homologues (ie, H-, K-, and N-Ras) makes distinct contributions to cellular activation. We examined the expression of each Ras protein in synovial tissue and FLS obtained from patients with rheumatoid arthritis and other forms of inflammatory arthritis. Each Ras protein was expressed in synovial tissue and cultured FLS. Each homolog was also activated following FLS stimulation with tumor necrosis factor-α or interleukin (IL)-1ß. Constitutively active mutants of each Ras protein enhanced IL-1ß-induced FLS matrix metalloproteinase-3 production, while only active H-Ras enhanced IL-8 production. Gene silencing demonstrated that each Ras protein contributed to IL-1ß-dependent IL-6 production, while H-Ras and N-Ras supported IL-1ß-dependent matrix metalloproteinase-3 and IL-8 production, respectively. The overlap in contributions of Ras homologues to FLS activation suggests that broad targeting of Ras GTPases in vivo suppresses global inflammation and joint destruction in arthritis. Consistent with this, simultaneous silencing of H-Ras, K-Ras, and N-Ras expression significantly reduces inflammation and joint destruction in murine collagen-induced arthritis, while specific targeting of N-Ras alone is less effective in providing clinical benefits.