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.

'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.

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.

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.

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.

Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor α7 subunit gene knockout mice.

BACKGROUND: The alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the alpha7nAChR can reduce the severity of arthritis in murine collagen-induced arthritis (CIA). OBJECTIVE: To provide more insight into the role of the alpha7nAChR in the pathogenesis of arthritis by investigating the effect of the absence of alpha7nAChR in CIA in alpha7-deficient (alpha7nAChR(-/-)) compared with wild-type (WT) mice. METHODS: CIA was induced in alpha7nAChR(-/-) and WT littermate mice at day 0 by immunisation with chicken collagen type II (cCII) followed by a booster injection with cCII on day 20. Mice were killed on day 44 or day 63 and arthritis activity as well as radiological and histological damage were scored. The effects on the immune response were evaluated by measurement of antigen-specific antibodies and cytokines, and evaluation of the effects on antigen-specific stimulated spleen cells. RESULTS: In alpha7nAChR(-/-) mice a significant increase in the incidence and severity of arthritis as well as increased synovial inflammation and joint destruction were seen. Exacerbation of CIA was associated with elevated systemic proinflammatory cytokines and enhanced T-helper cell 1 (Th1)-cytokine and tumour necrosis factor alpha production by spleen cells. Moreover, a specific decrease in the collagen-specific 'Th1-associated' IgG2a response was seen, whereas IgG1 titres were unaffected. CONCLUSIONS: The results presented here indicate that immune cell function in a model of rheumatoid arthritis is regulated by the cholinergic system and, at least in part, mediated by the alpha7nAChR.

Adeno-associated virus type 5-mediated intraarticular administration of tumor necrosis factor small interfering RNA improves collagen-induced arthritis.

OBJECTIVE: RNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated virus type 5 (rAAV5) for optimized intraarticular gene transfer, we undertook the present study to determine the feasibility of using rAAV5-mediated RNAi-based therapy in arthritis. METHODS: We developed rAAV5 vectors expressing short hairpin small interfering RNA (shRNA) against tumor necrosis factor alpha (TNFalpha) under H1 promoter, and carrying the enhanced green fluorescent protein (eGFP) reporter gene under cytomegalovirus promoter (rAAV5-shTNF). TNFalpha gene silencing was validated in vitro with mouse macrophages. Mice with collagen-induced arthritis were injected in the ankle and knee joints, at disease onset, with either rAAV5-shTNF or control rAAV5-eGFP vectors (5 x 10(9) particles). Arthritis severity was assessed clinically and histologically, and immunologic response was examined. Local and systemic transgene expression was monitored using quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, and enzyme-linked immunosorbent assay. RESULTS: After a single injection of rAAV5-shTNF into inflamed joints, local TNFalpha gene silencing provided rapid and long-term suppression of arthritis progression and reduced joint damage compared with that observed in control groups. Treatment with rAAV5-shTNF was associated with decreased proliferation and interferon-gamma production by antigen-stimulated T cells from draining lymph nodes, and the potency of this treatment was similar to that observed with other treatment strategies targeting TNFalpha at the protein level, either locally or systemically. CONCLUSION: Our data present the first proof-of-concept for the application of rAAV5-mediated RNAi-based gene therapy for local blockade of inflammation in experimental arthritis.

A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis.

INTRODUCTION: The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests. RESULTS: Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor alpha, interferon gamma and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help. CONCLUSIONS: The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.

Gene therapy targeting nuclear factor-kappaB: towards clinical application in inflammatory diseases and cancer.

Nuclear factor (NF)-kappaB is regarded as one of the most important transcription factors and plays an essential role in the transcriptional activation of pro-inflammatory cytokines, cell proliferation and survival. NF-kappaB can be activated via two distinct NF-kappaB signal transduction pathways, the so-called canonical and non-canonical pathways, and has been demonstrated to play a key role in a wide range of inflammatory diseases and various types of cancer. Much effort has been put in strategies to inhibit NF-kappaB activation, for example by the development of pharmacological compounds that selectively inhibit NF-kappaB activity and therefore would be beneficial for immunotherapy of transplantation, autoimmune and allergic diseases, as well as an adjuvant approach in patients treated with chemotherapy for cancer. Gene therapy targeting NF-kappaB is a promising new strategy with the potential of long-term effects and has been explored in a wide variety of diseases, ranging from cancer to transplantation medicine and autoimmune diseases. In this review we discuss recent progress made in the development of NF-kappaB targeted gene therapy and the evolution towards clinical application.

The alpha7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: a possible role for a key neurotransmitter in synovial inflammation.

OBJECTIVE: Recent studies have suggested an important role for neurotransmitters as modulators of inflammation. Therefore, we undertook this study to investigate the expression of the alpha7 subunit of the nicotinic acetylcholine receptor (alpha7nAChR) and its function in rheumatoid arthritis (RA). METHODS: The potential role of the alpha7nAChR in modulating proinflammatory cytokine expression in fibroblast-like synoviocytes (FLS) was identified by screening an adenoviral short hairpin RNA (Ad.shRNA) library. An alpha7-specific antibody was used for immunohistochemistry, and fluorescein isothiocyanate-labeled alpha-bungarotoxin, which binds specifically to the alpha7nAChR, was used for immunofluorescence. Gene expression in FLS was determined by quantitative polymerase chain reaction with primers specific for the alpha7nAChR. In addition, we analyzed messenger RNA (mRNA) expression of dupalpha7, a variant alpha7 transcript. Next, we studied the functional role of the alpha7nAChR in RA FLS by examining the effects of alpha7-specific agonists on the production of interleukin-6 (IL-6) and IL-8 by activated FLS. RESULTS: A screen using an Ad.shRNA library against 807 transcripts revealed that a specific alpha7nAChR shRNA potently modulated IL-8 and matrix metalloproteinase expression in FLS. The alpha7nAChR was expressed in the inflamed synovium from RA patients, predominantly in the intimal lining layer. We found alpha7nAChR expression at both the mRNA and protein level in cultured RA FLS. FLS also constitutively expressed dupalpha7 mRNA. Specific alpha7nAChR agonists reduced tumor necrosis factor alpha-induced IL-6 and IL-8 production by FLS. CONCLUSION: The alpha7nAChR and its dupalpha7 variant are expressed in RA synovium, where they may play a critical role in regulating inflammation. Targeting the alpha7nAChR could provide a novel antiinflammatory approach to the treatment of RA.

Stimulation of nicotinic acetylcholine receptors attenuates collagen-induced arthritis in mice.

OBJECTIVE: The parasympathetic nervous system, through the vagus nerve, can down-regulate inflammation in vivo by decreasing the release of cytokines, including tumor necrosis factor alpha (TNFalpha), by activated macrophages. The vagus nerve may exert antiinflammatory actions via a specific effect of its principal neurotransmitter, acetylcholine, on the alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) on macrophages. The present study was undertaken to obtain insight into the role of the cholinergic antiinflammatory pathway in arthritis. METHODS: To inhibit the cholinergic antiinflammatory pathway, mice were subjected to unilateral cervical vagotomy or sham surgery, after which arthritis was induced with type II collagen. In a separate study, nicotine was added to the drinking water of mice with collagen-induced arthritis (CIA). In addition, we investigated the effects of intraperitoneally (IP)-injected nicotine and the specific alpha7nAChR agonist AR-R17779. RESULTS: Clinical arthritis was exacerbated by vagotomy and ameliorated by oral nicotine administration. Moreover, oral nicotine inhibited bone degradation and reduced TNFalpha expression in synovial tissue. Both IP-injected nicotine and AR-R17779 ameliorated clinical arthritis and reduced synovial inflammation. This was accompanied by a reduction of TNFalpha levels in both plasma and synovial tissue. The effect of AR-R17779 was more potent compared with that of nicotine and was associated with delayed onset of the disease as well as with protection against joint destruction. CONCLUSION: These data provide the first evidence of a role of the cholinergic antiinflammatory pathway in the murine CIA model of rheumatoid arthritis.