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Significance Fibrosis remains a major unmet medical need as therapeutic targets discovered in animal models have failed to translate. A major challenge for identifying novel targets is the limited availability of early-stage human disease tissue. Here, we utilize Dupuytren’s disease (DD), a common localized fibrotic disorder, to evaluate the impact of epigenetic regulation of myofibroblasts and identify potential tractable targets in human fibrosis. We demonstrate that the epigenetic regulator CREBBP/EP300 is a critical determinant of the profibrotic phenotype. Furthermore, we identify collagen VI to be a key downstream target of CREBBP/EP300 and reveal valuable insights in the role it plays in key profibrotic functions, including contractile force, chemotaxis, and wound healing, and hence its potential as a therapeutic target., Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren’s disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, ACTA2 and COL1A1. Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren’s nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders.

Interest in CBG (cannabigerol) has been growing in the past few years, due to its anti-inflammatory properties and other therapeutic benefits. Here we report the synthesis of three new CBG derivatives (HUM-223, HUM-233 and HUM-234) and show them to possess anti-inflammatory and analgesic properties. In addition, unlike CBG, HUM-234 also prevents obesity in mice fed a high-fat diet (HFD). The metabolic state of the treated mice on HFD is significantly better than that of vehicle-treated mice, and their liver slices show significantly less steatosis than untreated HFD or CBG-treated ones from HFD mice. We believe that HUM-223, HUM-233 and HUM-234 have the potential for development as novel drug candidates for the treatment of inflammatory conditions, and in the case of HUM-234, potentially for obesity where there is a huge unmet need.

Background and Aims End stage kidney disease (ESKD) is associated with a malnutrition-inflammation complex that results in several endocrine, musculoskeletal, and metabolic abnormalities. Despite the known resultant derangements from this process, few studies have evaluated the associations between inflammation and skeletal status in ESKD patients. Our goal is to evaluate correlations between inflammatory and biochemical bone profile within and between dialysis patients and a healthy cohort. Method This analysis is a cross-sectional evaluation of a cohort of 40 dialysis patients and 20 healthy controls. Results Significant differences were found between the dialysis and healthy cohort in biochemical bone profile, inflammatory markers and adipokine levels. Dialysis patients with low vs. high PTH (cutoff 50 pg/mL) had differences in bone turnover markers but no differences in inflammatory or adipokine levels (Table 1). More specifically, bone-specific alkaline phosphatase (BSAP), osteocalcin (OCN) and osteoprotegerin (OPG) were positively correlated with insulin-like growth factors but this correlation was not present after adjustment for parathyroid hormone (PTH) levels. C-terminal cross-linking telopeptide (CTX) positively correlated with IGF1 (p-value 0.02, 95% CI (0.14-1.69)) and with IGFBP3 (p-value 0.007 and 95% CI (0.31- 1.84)) after multivariate adjustment for PTH, albumin and sex. Adiponectin positively correlated with IGFBP3 and this remained significant after adjustment for PTH, albumin and sex (p=0.02). IL6, IL8 and TNF did not correlate with bone turnover markers or with adipokine levels after multivariate adjustments. Conclusion In this cross-sectional study, significant differences were found between the dialysis and healthy cohort in biochemical bone profile, inflammatory markers and adipokine levels. In the dialysis cohort, inflammatory markers did not correlate with bone turnover markers or with adipokine levels in dialysis patients. Dialysis patients with high vs. low PTH (cutoff 50 pg/mL) had higher levels of bone formation and bone resorption markers. Inflammatory and adipokine levels did not differ between those with high vs. low PTH.

Fibrotic disorders are some of the most devastating and poorly treated conditions in developed nations, yet effective therapeutics are not identified for many of them. A major barrier for the identification of targets and successful clinical translation is a limited understanding of the human fibrotic microenvironment. Here, we construct a stromal cell atlas of human fibrosis at single cell resolution from patients with Dupuytren’s disease, a localized fibrotic condition of the hand. A molecular taxonomy of the fibrotic milieu characterises functionally distinct stromal cell types and states, including a subset of immune regulatory ICAM1+ fibroblasts. In developing fibrosis, myofibroblasts exist along an activation continuum of phenotypically distinct populations. We also show that the tetraspanin CD82 regulates cell cycle progression and can be used as a cell surface marker of myofibroblasts. These findings have important implications for targeting core pathogenic drivers of human fibrosis., New anti-fibrotics aided by a better understanding of fibrotic cell subsets and their functions are needed. Here the authors perform single-cell RNA-seq and CyTOF on nodules from patients with Dupuytren’s disease to map subsets and the state of mesenchymal cells during pathogenic fibrogenesis.

Fibrotic disorders are some of the most devastating and poorly treated conditions in developed nations, yet effective therapeutics are not identified for many of them. A major barrier for the identification of targets and successful clinical translation is a limited understanding of the human fibrotic microenvironment. Here, we construct a stromal cell atlas of human fibrosis at single cell resolution from patients with Dupuytren’s disease, a localized fibrotic condition of the hand. A molecular taxonomy of the fibrotic milieu characterises functionally distinct stromal cell types and states, including a subset of immune regulatory ICAM1+ fibroblasts. In developing fibrosis, myofibroblasts exist along an activation continuum of phenotypically distinct populations. We also show that the tetraspanin CD82 regulates cell cycle progression and can be used as a cell surface marker of myofibroblasts. These findings have important implications for targeting core pathogenic drivers of human fibrosis. New anti-fibrotics aided by a better understanding of fibrotic cell subsets and their functions are needed. Here the authors perform single-cell RNA-seq and CyTOF on nodules from patients with Dupuytren’s disease to map subsets and the state of mesenchymal cells during pathogenic fibrogenesis.

IL-33 mediates cross-talk between immune and stromal cells in localized fibrosis., Dissecting the molecular landscape of fibrotic disease, a major unmet need, will inform the development of novel treatment strategies to target disease progression and identify desperately needed therapeutic targets. Here, we provide a detailed single-cell analysis of the immune landscape in Dupuytren’s disease, a localized fibrotic condition of the hand, and identify a pathogenic signaling circuit between stromal and immune cells. We demonstrate M2 macrophages and mast cells as key cellular sources of tumor necrosis factor (TNF) that promotes myofibroblast development. TNF acts via the inducible TNFR2 receptor and stimulates interleukin-33 (IL-33) secretion by myofibroblasts. In turn, stromal cell IL-33 acts as a potent stimulus for TNF production from immune cells. Targeting this reciprocal signaling pathway represents a novel therapeutic strategy to inhibit the low-grade inflammation in fibrosis and the mechanism that drives chronicity.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 play an important role in maintaining immune homeostasis. Chronic inflammation is associated with reduced Foxp3 expression, function, and loss of phenotypic stability. Previous studies have established the importance of TNF receptor 2 (TNFR2) in the generation and/or activation of Treg cells. In this study, we assess the importance of TNFR2 in healthy mice and under inflammatory conditions. Our findings reveal that, in health, TNFR2 is important not only for the generation of Treg cells, but also for regulating their functional activity. We also show that TNFR2 maintains Foxp3 expression in Treg cells by restricting DNA methylation at the Foxp3 promoter. In inflammation, loss of TNFR2 results in increased severity and chronicity of experimental arthritis, reduced total numbers of Treg cells, reduced accumulation of Treg cells in inflamed joints, and loss of inhibitory activity. In addition, we demonstrate that, under inflammatory conditions, loss of TNFR2 causes Treg cells to adopt a proinflammatory Th17-like phenotype. It was concluded that TNFR2 signaling is required to enable Treg cells to promote resolution of inflammation and prevent them from undergoing dedifferentiation. Consequently, TNFR2-specific agonists or TNF1-specific antagonists may be useful in the treatment of autoimmune disease.

Tumour necrosis factor-α (TNF-α) is a highly pleiotropic cytokine with effects on multiple pathological and physiological functions via two distinct receptors, TNFR1 and TNFR2. Much of the pro- inflammatory action of TNF-α is mediated by TNFR1 whereas TNFR2 is thought to play an immunoregulatory and tissue protective role. Anti-TNF- α biologics have been extremely successful in treating a number of immune mediated pathologies, including rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis and inflammatory bowel disease. However, anti-TNF therapy has been shown to induce systemic lupus erythematosus and psoriasis in some patients, and to be deleterious in multiple sclerosis. It is hypothesized that these paradoxical effects of anti-TNF-α are due to inhibition of TNFR2 signalling. In this review, we will focus on the biology and pathophysiologic role of TNF-α and on the therapeutic implications of targeting TNF-α receptor signalling.

Chemical probes are small molecules with potency and selectivity for a single or small number of protein targets. A good chemical probe engages its target intracellularly and is accompanied by a chemically similar, but inactive molecule to be used as a negative control in cellular phenotypic screening. The utility of these chemical probes is ultimately governed by how well they are developed and characterized. Chemical probes either as single entities, or in chemical probes sets are being increasingly used to interrogate the biological relevance of a target in a disease model. This chapter lays out the core properties of chemical probes, summarizes the seminal and emerging techniques used to demonstrate robust intracellular target engagement. Translation of target engagement assays to disease-relevant phenotypic assays using primary patient-derived cells and tissues is also reviewed. Two examples of epigenetic chemical probe discovery and utility are presented whereby target engagement pointed to novel disease associations elucidated from poorly understood protein targets. Finally, a number of examples are discussed whereby chemical probe sets, or "chemogenomic libraries" are used to illuminate new target-disease links which may represent future directions for chemical probe utility.

Animal models are used for the study of a number of human autoimmune diseases, including multiple sclerosis, diabetes, rheumatoid arthritis, systemic lupus erythematosis and spondyloarthropathies. Induced, spontaneous and genetically manipulated animal models can be described in terms of their parallels to human disease and as valuable tools for the development of potential therapies. Studies in animal models have led to a number of important discoveries, which have increased our understanding of the pathogenesis of autoimmune disease, including the roles played by regulatory T cells and TH17 cells. In addition, important therapeutic advances have emerged as a result of studies of immune intervention in animal models of autoimmunity. For example, tumour necrosis factor (TNF) blocking drugs, which are widely used for the treatment of rheumatic diseases, were developed following pre-clinical testing in animal models. Key Concepts Animal models may be either spontaneously occurring or induced as a result of genetic manipulation, immunisation with a self-antigen or triggered by pathogen-associated molecular patterns (PAMPs) in genetically susceptible hosts. No animal model completely mimics human disease. Animal models can be used to delineate common mammalian immunological mechanisms, test novel therapeutic concepts and understand mechanisms of drug action, but therapeutic efficacy therein is not predictive of response in clinical trials. The use of transgenic and knockout strains facilitates the identification of key genes that contribute to disease susceptibility and pathogenesis. Keywords: animal models; rheumatoid arthritis; spondyloarthropathy; ankylosing spondylitis; collagen-induced arthritis; SKG

Background Despite the overall success of TNFα inhibitors in rheumatoid arthritis (RA), up to half of patients are classified as either primary or secondary non-responders1. One hypothesis put forward to explain resistance to anti-TNFα therapy is an ascendant effect of dysregulated regulatory T cells and increased Th17 responses following TNFα blockade. Previous studies have demonstrated that TNFR2 is critical for stabilisation and suppressive function of regulatory T cells2,3. However, TNFR2 also activates pro-inflammatory signalling cascades and, to date, the net effect of TNFR2 on the pathogenesis of RA remains unclear. Objectives In this study we address this question by assessing the progression of collagen-induced arthritis (CIA) in mice deficient for TNFR1 or TNFR2. Methods C57Bl/6N.Q (H-2q) mice were immunised with bovine type II collagen emulsified in complete Freund’s adjuvant. The mice were monitored daily for arthritis and scored clinically from the day of onset of disease. Mice were culled on day 10 after arthritis onset and spleens, lymph nodes, serum and paws were collected for further analysis. Results As expected, TNFR1-/- mice were found to be largely resistant to arthritis both clinically and histologically (figure 1). In contrast, there was significantly enhanced disease activity at the clinical and histological levels in TNFR2-/- mice (figure 1) and this was accompanied by increased expression of the pro-inflammatory cytokines, TNFα and IL-6, reduced numbers of regulatory T cells, reduced FoxP3 expression and reduced expression of the immune inhibitory molecules, PD-1 and LAG3, in TNFR2-/- mice compared to WT mice. Conclusions This study has shown that TNFR2 signalling plays immunoregulatory and anti-inflammatory roles in CIA. First, it contributes to promotion of regulatory T cell generation and FoxP3 expression, and second, it limits the expression of pro-inflammatory cytokines. TNFR2 also regulates the expression immune inhibitory molecules during inflammation. The results support the rationale to for development of TNFR1 specific antagonists or TNFR2 agonists for the treatment of RA. References [1] Giulia Roda, et al. Loss of Response to Anti-TNFs: Definition, Epidemiology, and Management. Clinical and Translational Gastroenterology2016. [2] Xin Chen, et al. TNFR2 is critical for the stabilization of the CD4+Foxp3+ regulatory T cell phenotype in the inflammatory enviroment. J. Immunol2013. [3] Xin Chen et al. Expression of TNFR2 defines a maximally suppressive subset of mouse CD4 +CD25+FoxP3+T regulatory cells: applicability to tumour infiltrating T regulatory cells. J. Immunology 2008. Acknowledgements This work was supported by grants from Chang Gung Memorial Hospital and Ministry of Science and Technology (Taiwan) Disclosure of Interest None declared

Chemical probes are small molecules with potency and selectivity for a single or small number of protein targets. A good chemical probe engages its target intracellularly and is accompanied by a chemically similar, but inactive molecule to be used as a negative control in cellular phenotypic screening. The utility of these chemical probes is ultimately governed by how well they are developed and characterized. Chemical probes either as single entities, or in chemical probes sets are being increasingly used to interrogate the biological relevance of a target in a disease model. This chapter lays out the core properties of chemical probes, summarizes the seminal and emerging techniques used to demonstrate robust intracellular target engagement. Translation of target engagement assays to disease-relevant phenotypic assays using primary patient-derived cells and tissues is also reviewed. Two examples of epigenetic chemical probe discovery and utility are presented whereby target engagement pointed to novel disease associations elucidated from poorly understood protein targets. Finally, a number of examples are discussed whereby chemical probe sets, or "chemogenomic libraries" are used to illuminate new target-disease links which may represent future directions for chemical probe utility.

Pattern recognition underpins innate immunity; the accurate identification of danger, including infection, injury, or tumor, is key to an appropriately targeted immune response. Pathogen detection is increasingly well defined mechanistically, but the discrimination of endogenous inflammatory triggers remains unclear. Tenascin-C, a matrix protein induced upon tissue damage and expressed by tumors, activates toll-like receptor 4 (TLR4)-mediated sterile inflammation. Here we map three sites within tenascin-C that directly and cooperatively interact with TLR4. We also identify a conserved inflammatory epitope in related proteins from diverse families, and demonstrate that its presence targets molecules for TLR detection, while its absence enables escape of innate immune surveillance. These data reveal a unique molecular code that defines endogenous proteins as inflammatory stimuli by marking them for recognition by TLRs., Although detection of pathogens by pattern recognition receptors is increasingly well defined, recognition of endogenous triggers remains poorly understood. By examining the interface between tenascin-C and TLR4, the authors identify a molecular code that identifies endogenous proteins as inflammatory stimuli.

Blazek et al. demonstrate that treatment with IL-28A reduces inflammation in collagen-induced arthritis by restricting the recruitment of IL-1β+ neutrophils., The most studied biological role of type III interferons (IFNs) has so far been their antiviral activity, but their role in autoimmune and inflammatory diseases remains largely unexplored. Here, we show that treatment with IFN-λ2/IL-28A completely halts and reverses the development of collagen-induced arthritis (CIA) and discover cellular and molecular mechanisms of IL-28A antiinflammatory function. We demonstrate that treatment with IL-28A dramatically reduces numbers of proinflammatory IL-17–producing Th17 and γδ T cells in the joints and inguinal lymph nodes, without affecting T cell proliferative responses or levels of anticollagen antibodies. IL-28A exerts its antiinflammatory effect by restricting recruitment of IL-1b–expressing neutrophils, which are important for amplification of inflammation. We identify neutrophils as cells expressing high levels of IFN-λ receptor 1 (IFNLR1)–IL-28 receptor α (IL28RA) and targeted by IL-28A. Our data highlight neutrophils as contributors to the pathogenesis of autoimmune arthritis and present IFN-λs or agonists of IFNLR1–IL28RA as putative new therapeutics for neutrophil-driven inflammation.

Objective Tumor necrosis factor (TNF) signals via 2 receptors, TNFR type I (TNFRI) and TNFRII, with distinct cellular distribution and signaling functions. In rheumatoid arthritis (RA), the net effect of TNFR signaling favors inflammatory responses while inhibiting the activity of regulatory T cells. TNFRII signaling has been shown to promote Treg cell function. To assess the relative contributions of TNFRI and TNFRII signaling to inflammatory and regulatory responses in vivo, we compared the effect of TNF blockade, hence TNFRI/II, versus TNFRI alone in collagen-induced arthritis (CIA) as a model of RA. Methods Mice with established arthritis were treated for 10 days with anti-mouse TNFRI domain antibody (dAb; DMS5540), an isotype control dAb (DMS5538), or murine TNFRII genetically fused with mouse IgG1 Fc domain (mTNFRII-Fc) beginning on the day of arthritis onset, and disease progression was monitored. Systemic cytokine concentrations and numbers of T cell subsets in lymph nodes and spleens were measured, and intrinsic Treg cell function was determined by ex vivo suppression assays. Results Progression of CIA was suppressed similarly by TNFRI (DMS5540) and TNFRI/II (mTNFRII-Fc) blockade. However, blockade of TNFRI/II led to increased effector T cell activity, which was not observed after selective TNFRI blockade, suggesting an immunoregulatory role of TNFRII. In support of this, TNFRI blockade, but not TNFRI/II blockade, expanded and activated Treg cells. Furthermore, a dramatic increase in expression of the Treg cell signature genes FoxP3 and TNFRII was observed in joints undergoing remission, which supports the notion that these molecules have a physiologic role in the resolution of inflammation. Conclusion We propose that a therapeutic strategy that targets TNFRI while sparing TNFRII has the potential to both inhibit inflammation and promote Treg cell activity, which might be superior to TNF blockade.

An immune response is essential for protection against infection, but, in many individuals, aberrant responses against self tissues cause autoimmune diseases such as rheumatoid arthritis (RA). How to diminish the autoimmune response while not augmenting infectious risk is a challenge. Modern targeted therapies such as anti-TNF or anti-CD20 antibodies ameliorate disease, but at the cost of some increase in infectious risk. Approaches that might specifically reduce autoimmunity and tissue damage without infectious risk would be important. Here we describe that TNF superfamily member OX40 ligand (OX40L; CD252), which is expressed predominantly on antigen-presenting cells, and its receptor OX40 (on activated T cells), are restricted to the inflamed joint in arthritis in mice with collagen-induced arthritis and humans with RA. Blockade of this pathway in arthritic mice reduced inflammation and restored tissue integrity predominantly by inhibiting inflammatory cytokine production by OX40L-expressing macrophages. Furthermore, we identify a previously unknown role for OX40L in steady-state bone homeostasis. This work shows that more targeted approaches may augment the "therapeutic window" and increase the benefit/risk in RA, and possibly other autoimmune diseases, and are thus worth testing in humans.

Fluorescence imaging is used widely in microscopy and macroscopic imaging applications for fields ranging from biomedicine to materials science. A critical component for any fluorescence imaging system is the excitation source. Traditionally, wide-field systems use filtered thermal or arc-generated white light sources, while point scanning confocal microscope systems require spatially coherent (point-like) laser sources. Unfortunately, the limited range of visible wavelengths available from conventional laser sources constrains the design and usefulness of fluorescent probes in confocal microscopy. A 'hands-off ' laser-like source, electronically tunable across the visible spectrum, would be invaluable for fluorescence imaging and provide new opportunities, e.g. automated excitation fingerprinting and in situ measurement of excitation cross-sections. Yet more information can be obtained using fluorescence lifetime imaging (FLIM), which requires that the light source be pulsed or rapidly modulated. We show how a white light continuum, generated by injecting femtosecond optical radiation into a micro-structured optical fibre, coupled with a simple prism-based tunable filter arrangement, can fulfil all these roles as a continuously electronically tunable (435-1150 nm) visible ultrafast light source in confocal, wide-field and FLIM systems.

As for any disease, unravelling the underlying mechanisms involved in the pathogenicity of rheumatoid arthritis is key to providing clues for designing effective therapeutic strategies that can ultimately translate from the laboratory o the clinic. Here, we provide an overview of rheumatoid arthritis and discuss experimental evidence suggesting how genetic and environmental factors, such as MHC genes, smoking and female sex hormones, can contribute to disease susceptibility. In established disease, the role of T cells is discussed in light of increased numbers of T cells in the arthritic versus healthy joint and the observed therapeutic effects of T cell modifying agents such as anti-CD4, anti-TCR and anti-IL2R antibodies in animal models of rheumatoid arthritis. As a vital tool, crucial to furthering our understanding of disease mechanisms, we elaborate on various animal models of rheumatoid arthritis being extensively exploited in arthritis research, including adjuvant-induced arthritis, collagen-induced arthritis, and transgenic mice expressing TNFα or IL-1β genes. Such experimental disease models have led to significant discoveries relating to the importance of pro-inflammatory cytokines in the pathogenesis of rheumatoid arthritis, resulting from a disregulation of the normally finely tuned balance of pro- and anti-inflammatory cytokine signalling. Indeed, the most successful therapeutic to date for rheumatoid arthritis, anti-TNFα, was initially studied in collagen-induced arthritis. It is concluded that mechanistic based research is essential to elucidate and evaluate new candidate therapies for the treatment of rheumatoid arthritis, and other autoimmune diseases. © 2008 Bentham Science Publishers Ltd.

T cell–APC conjugation as mediated by leukocyte function-associated antigen-1 (LFA-1)–intercellular adhesion molecule (ICAM)-1 binding is followed by formation of the supramolecular activation cluster (SMAC) at the immunological synapse. The intracellular processes that regulate SMAC formation and its influence on T cell function are important questions to be addressed. Here, using a mutational approach, we demonstrate that binding of adaptor adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76 differentially regulates peripheral SMAC (pSMAC) formation relative to conjugation. Although mutation of the YDDV sites (termed M12) disrupted SLP-76 SH2 domain binding and prevented the ability of ADAP to increase conjugation and LFA-1 clustering, M12 acted selectively as a dominant negative (DN) inhibitor of pSMAC formation, an effect that was paralleled by a DN effect on interleukin-2 production. ADAP also colocalized with LFA-1 at the immunological synapse. Our findings identify ADAP–SLP-76 binding as a signaling event that differentially regulates SMAC formation, and support a role for SMAC formation in T cell cytokine production.

Heterogeneity in the supramolecular organization of immunological synapses arises from the involvement of different cells, distinct environmental stimuli, and varying levels of protein expression. There may also be heterogeneity in the types and amounts of cell surface proteins and lipids that transfer between lymphocytes during immune surveillance. In addition, immune cells can be involved in the assembly of a ‘viral synapse’, such that micrometer-scale organization of proteins at intercellular contacts occurs during transmission of a virus between T cells. Thus, while there may be unity in molecular mechanisms underlying the organization of cell surface receptors at immune cell synapses, there is diversity in their function.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation leading to tissue destruction and progressive loss of joint function. Here we describe two methods that can be used to assess the contribution of toll-like receptors (TLRs), and their potential ligands, to RA pathogenesis. We focus on the antigen-induced model of murine arthritis and human synovial tissue explant models. Both enable detection of TLR, and TLR ligand, expression, as well as investigation of the effect of inhibition of these molecules. Each offers a unique insight into disease; with murine models allowing kinetic analysis in live animals and explant models allowing examination of inflamed human tissue, which together can help us to dissect the role of TLRs in the onset and progression of RA.

Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation leading to tissue destruction and progressive loss of joint function. Here we describe two methods that can be used to assess the contribution of toll-like receptors (TLRs), and their potential ligands, to RA pathogenesis. We focus on the antigen-induced model of murine arthritis and human synovial tissue explant models. Both enable detection of TLR, and TLR ligand, expression, as well as investigation of the effect of inhibition of these molecules. Each offers a unique insight into disease; with murine models allowing kinetic analysis in live animals and explant models allowing examination of inflamed human tissue, which together can help us to dissect the role of TLRs in the onset and progression of RA.

Objective To assess the therapeutic potential of anti-CD3 monoclonal antibodies (mAb) for rheumatoid arthritis, using collagen-induced arthritis as an animal model. Methods Arthritis was induced in DBA/1 mice by immunization with type II collagen. After disease onset, a single injection of anti-CD3 mAb (20 μg/mouse) was administered, and arthritis severity was monitored over a 10-day period. Results Anti-CD3 mAb treatment resulted in a sustained reduction in disease activity, which was associated with an increase in the proportion of naturally occurring CD4+CD25+FoxP3+ regulatory T (Treg) cells and the generation of a population of CD8+CD25+FoxP3+ Treg cells. Anti-CD3 mAb treatment did not alter the capacity of CD4+ Treg cells to suppress effector T cell proliferation and interferon-γ (IFNγ) production in vitro. However, CD4+ Treg cells from both anti-CD3 mAb–treated and control mice were unable to suppress interleukin-17 (IL-17) production. In contrast, CD8+ Treg cells induced by anti-CD3 therapy suppressed IL-17 production as well as CD4+ T cell proliferation and IFNγ production. Conclusion These results show that anti-CD3 mAb treatment has important therapeutic potential for rheumatoid arthritis and has the capacity to generate antiarthritic CD8+ Treg cells and expand the relative numbers of CD4+ Treg cells.

Intercellular transfer of cell surface proteins is widespread and facilitates several recently discovered means for immune cell communication. Here, we examined the molecular mechanism for intercellular exchange of the natural killer (NK) cell receptor KIR2DL1 and HLA-C, prototypical proteins that swap between NK cells and target cells. Transfer was contact dependent and enhanced for cells expressing cognate receptor/ligand pairs but did not depend on KIR2DL1 signaling. To a lesser extent, proteins transferred independent from specific recognition. Intracellular domains of transferred proteins were not exposed to the extracellular environment and transferred proteins were removed by brief exposure to low pH. By fluorescence microscopy, transferred proteins localized to discrete regions on the recipient cell surface. Higher resolution scanning electron micrographs revealed that transferred proteins were located within specific membranous structures. Transmission electron microscopy of the immune synapse revealed that membrane protrusions from one cell interacted with the apposing cell surface within the synaptic cleft. These data, coupled with previous observations, lead us to propose that intercellular protein transfer is mediated by membrane protrusions within and surrounding the immunological synapse.

NKG2D is an activating receptor expressed on all human NK cells and a subset of T cells. In cytolytic conjugates between NK cells and target cells expressing its ligand MHC class I chain-related gene A, NKG2D accumulates at the immunological synapse with GM1-rich microdomains. Furthermore, NKG2D is specifically recruited to detergent-resistant membrane fractions upon ligation. However, in the presence of a strong inhibitory stimulus, NKG2D-mediated cytotoxicity can be intercepted, and recruitment of NKG2D to the immunological synapse and detergent-resistant membrane fractions is blocked. Also, downstream phosphorylation of Vav-1 triggered by NKG2D ligation is circumvented by coengaging inhibitory receptors. Thus, we propose that one way in which inhibitory signaling can control NKG2D-mediated activation is by blocking its recruitment to GM1-rich membrane domains. The accumulation of activating NK cell receptors in GM1-rich microdomains may provide the necessary platform from which stimulatory signals can proceed.

Supramolecular clusters at the immunological synapse provide a mechanism for structuring complex communication networks between cells of the immune system. Regulating intra- and intercellular trafficking of proteins and lipids to and from the immunological synapse provides an additional level of complexity in determining the functional outcome of immune cell interactions. An emergent principle is that molecules requiring tightly regulated cell surface expression, e.g. negative regulators of cell activation or molecules promoting cytotoxicity, are trafficked to the immunological synapse from intracellular secretory as required lysosomes. Many molecules required for the early stages of the intercellular communication are already present at the cell surface, sometimes in lipid rafts, and are rapidly translocated laterally to the intercellular contact. Our understanding of these events critically depends on utilizing appropriate technologies for probing supramolecular recognition in live cells. Thus, we also present here a critical discussion of the technologies used to study lipid rafts and, more broadly, a map of the spatial and temporal dimensions covered by current live cell physical techniques, highlighting where advances are needed to exceed current spatial and temporal boundaries.

In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS. This distribution of ezrin and CD43 at the inhibitory NK cell IS is similar to that previously seen at the activating T cell IS. CD45 is also excluded from the inhibitory, but not activating, NK cell IS. In addition, electron microscopy reveals wide and narrow domains across the synaptic cleft. Target cell HLA-C, located by immunogold labeling, clusters where the synaptic cleft spans the size of HLA-C bound to the inhibitory killer Ig-like receptor. These data are consistent with assembly of the NK cell IS involving a combination of cytoskeletal-driven mechanisms and thermodynamics favoring the organization of receptor/ligand pairs according to the size of their extracellular domains.

As T cells and natural killer (NK) cells survey the surface of other cells, cognate receptors and ligands are commonly organized into distinct micrometer-scale domains at the intercellular contact, creating an immune or immunological synapse (IS). We aim to address the still unanswered questions of how this organization of proteins aids immune surveillance and how these domains are biophysically constructed. Molecular mechanisms for the formation of the IS include a role for the cytoskeleton, segregation of proteins according to the size of their extracellular domains, and association of proteins with lipid rafts. Towards understanding the function of the IS, it is instructive to compare and contrast the supramolecular organization of proteins at the inhibitory and activating NK cell IS with that at the activating T cell IS. Finally, it is essential to develop new technologies for probing molecular recognition at cell surfaces. Imaging parameters other than fluorescence intensity, such as the lifetime of the fluorophore's excited state, could be used to report on protein environments.

Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2(-/-) macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NF-kappaB pathways was blocked in TNFR1(-/-) macrophages. In contrast, although TNFR2(-/-) macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB pro-inflammatory signalling pathways.

After accumulation of target cell human leukocyte antigen (HLA)-C at inhibitory natural killer (NK) cell immune synapses, some HLA-C transfers from target cells to NK cell plasma membranes and cytoplasm. This unexpected intercellular transfer of HLA-C is dependent on NK receptor recognition, since HLA-Cw6 or -Cw4 but not -Cw3 transfer to an NK transfectant expressing killer Ig-like receptor (KIR)2DL1. Strikingly, live-cell time-lapse laser scanning confocal microscopy shows vesicles containing target cell green fluorescent protein–tagged HLA-C migrating away from immune synapses into NK cells. Unlike clustering of HLA-C at the immune synapse, intercellular transfer of HLA-C is dependent on NK cell ATP, but not target cell ATP. However, the intercellular transfer of HLA-C is not dependent on active polymerization of the actin cytoskeleton. In addition, different arrangements of HLA-C are seen at inhibitory NK immune synapses, and these alter as NK synapses mature, but in a fashion distinct from that seen upon T cell activation.

Tumor necrosis factor (TNF) signals via 2 receptors, TNFR type I (TNFRI) and TNFRII, with distinct cellular distribution and signaling functions. In rheumatoid arthritis (RA), the net effect of TNFR signaling favors inflammatory responses while inhibiting the activity of regulatory T cells. TNFRII signaling has been shown to promote Treg cell function. To assess the relative contributions of TNFRI and TNFRII signaling to inflammatory and regulatory responses in vivo, we compared the effect of TNF blockade, hence TNFRI/II, versus TNFRI alone in collagen-induced arthritis (CIA) as a model of RA.Mice with established arthritis were treated for 10 days with anti-mouse TNFRI domain antibody (dAb; DMS5540), an isotype control dAb (DMS5538), or murine TNFRII genetically fused with mouse IgG1 Fc domain (mTNFRII-Fc) beginning on the day of arthritis onset, and disease progression was monitored. Systemic cytokine concentrations and numbers of T cell subsets in lymph nodes and spleens were measured, and intrinsic Treg cell function was determined by ex vivo suppression assays.Progression of CIA was suppressed similarly by TNFRI (DMS5540) and TNFRI/II (mTNFRII-Fc) blockade. However, blockade of TNFRI/II led to increased effector T cell activity, which was not observed after selective TNFRI blockade, suggesting an immunoregulatory role of TNFRII. In support of this, TNFRI blockade, but not TNFRI/II blockade, expanded and activated Treg cells. Furthermore, a dramatic increase in expression of the Treg cell signature genes FoxP3 and TNFRII was observed in joints undergoing remission, which supports the notion that these molecules have a physiologic role in the resolution of inflammation.We propose that a therapeutic strategy that targets TNFRI while sparing TNFRII has the potential to both inhibit inflammation and promote Treg cell activity, which might be superior to TNF blockade.

Psoriasis is a chronic inflammatory skin disease, most commonly resulting in the occurrence of red and silver scaly plaques. About 30% of psoriasis sufferers develop psoriatic arthritis (PsA), a disorder that presents with additional joint inflammation and other clinical features. At present, the most effective treatment for moderate and severe psoriasis and PsA are biologics such as antitumor necrosis factor alpha therapy. Biologics are costly and typically require repeated injections; hence, the development of novel, orally available, small molecular inhibitors that are less expensive to produce is highly desirable. The phosphodiesterase 4 inhibitor apremilast is a small molecular inhibitor that acts by increasing cyclic adenosine monophosphate levels, ultimately suppressing tumor necrosis alpha production. Apremilast has been tested in a number of psoriasis and PsA pilot and Phase II trials to evaluate its efficacy and safety. More recently, three larger double-blinded, and randomized multicenter studies demonstrate that apremilast is efficacious in the treatment of psoriasis and PsA, with significantly higher numbers of apremilast-treated patients achieving endpoints of a 75% reduction compared to baseline in Psoriasis Area and Severity Index (PASI-75) or American College of Rheumatology-20 scores, relative to placebo. This encouraging data, along with a tolerable incidence of mild to moderate adverse events, has led to the initiation of several large Phase III trials that aim to further validate apremilast as a treatment for psoriasis and PsA. Here, we provide an overview of the current treatments for psoriasis and PsA, and summarize the findings from multiple Phase II clinical trials where the effects of apremilast in the treatment of psoriasis and PsA patients have been investigated.

Objective To assess the role of the activating receptor NKG2D in arthritis. Methods Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, real-time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days. Results We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae-1 was up-regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down-modulated during the progression of collagen-induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti-NKG2D–treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti-NKG2D treatment significantly reduced both interleukin-17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro. Conclusion Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

IL-17 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by tumor necrosis factor (TNF) in vitro. The aim of this study was to assess the impact of TNF inhibition on IL-17 production in collagen-induced arthritis, a model of RA. TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic. Th1 and Th17 cell populations were also expanded in collagen-immunized TNFR p55−/− but not p75−/− mice. The expression of IL-12/IL-23 p40 was up-regulated in lymph nodes (LN) from p55−/− mice, and the expansion of Th1/Th17 cells was abrogated by blockade of p40. Treatment of macrophages with rTNF also inhibited p40 production in vitro. These findings indicate that at least one of the ways in which TNF regulates Th1/Th17 responses in arthritis is by down-regulating the expression of p40. Finally, although TNF blockade increased numbers of Th1 and Th17 cells in LN, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells.

We demonstrate a continuously electronically tunable (435-1150 nm) ultrafast source for fluorescence imaging applications that is derived from a visible supercontinuum generated by injecting infrared femtosecond pulses in to a microstructured fibre. We demonstrate this source applied to confocal and wide-field microscopy, as well as multiwell-plate imaging. We also report first application of a tunable supercontinuum source (TSS) to FLIM and to in situ measurements of relative excitation spectra. We are currently applying this source to hyperspectral imaging and working towards a system for acquiring the full fluorescence excitation-emission matrix. Fluorescence lifetime imaging (FLIM) can provide further information about fluorescent samples and requires a rapidly modulated or pulsed light source, in general, (tunable) ultrafast light sources, particularly in the visible, are complex and expensive and usually require a significant degree of adjustment to achieve tunability

Supramolecular clusters at the immunological synapse provide a mechanism for structuring complex communication networks between cells of the immune system. Regulating intra- and intercellular trafficking of proteins and lipids to and from the immunological synapse provides an additional level of complexity in determining the functional outcome of immune cell interactions. An emergent principle is that molecules requiring tightly regulated cell surface expression, e.g. negative regulators of cell activation or molecules promoting cytotoxicity, are trafficked to the immunological synapse from intracellular secretory as required lysosomes. Many molecules required for the early stages of the intercellular communication are already present at the cell surface, sometimes in lipid rafts, and are rapidly translocated laterally to the intercellular contact. Our understanding of these events critically depends on utilizing appropriate technologies for probing supramolecular recognition in live cells. Thus, we also present here a critical discussion of the technologies used to study lipid rafts and, more broadly, a map of the spatial and temporal dimensions covered by current live cell physical techniques, highlighting where advances are needed to exceed current spatial and temporal boundaries.

SummaryDietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine, with absorptive capacity decreasing distally. We utilized a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner. HCP1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP1 protein was iron regulated and localized to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP1 is the long-sought intestinal heme transporter.

Cell surface proteins major histocompatibility complex (MHC) class I–related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein–tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.

As T cells and natural killer (NK) cells survey the surface of other cells, cognate receptors and ligands are commonly organized into distinct micrometer-scale domains at the intercellular contact, creating an immune or immunological synapse (IS). We aim to address the still unanswered questions of how this organization of proteins aids immune surveillance and how these domains are biophysically constructed. Molecular mechanisms for the formation of the IS include a role for the cytoskeleton, segregation of proteins according to the size of their extracellular domains, and association of proteins with lipid rafts. Towards understanding the function of the IS, it is instructive to compare and contrast the supramolecular organization of proteins at the inhibitory and activating NK cell IS with that at the activating T cell IS. Finally, it is essential to develop new technologies for probing molecular recognition at cell surfaces. Imaging parameters other than fluorescence intensity, such as the lifetime of the fluorophore's excited state, could be used to report on protein environments.

INTRODUCTION: Type 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis. METHODS: Cells liberated from tissue excised from arthritic joints of RA patients were cultured in the presence of increasing concentrations of apremilast for 48 hours and spontaneous tumour necrosis factor-alpha (TNFalpha) production was analysed in culture supernatants by ELISA. In addition, arthritis was induced in BALB/c and DBA/1 mice by passive transfer of anti-type II collagen mAb and immunisation with type II collagen, respectively. Mice with established arthritis received 5 or 25 mg/kg apremilast and disease severity was monitored relative to mice receiving vehicle alone. At the end of the study, paws were removed and processed for histopathological assessment. Behavioural effects of apremilast, relative to rolipram, were assessed in naïve DBA/1 mice using an automated activity monitor (LABORAS). RESULTS: Apremilast dose dependently inhibited spontaneous release of TNFalpha from human rheumatoid synovial membrane cultures. Furthermore, apremilast significantly reduced clinical score in both murine models of arthritis over a ten day treatment period and maintained a healthy joint architecture in a dose-dependent manner. Importantly, unlike rolipram, apremilast demonstrated no adverse behavioural effects in naïve mice. CONCLUSIONS: Apremilast is an orally available PDE4 inhibitor that reduces TNFalpha production from human synovial cells and significantly suppresses experimental arthritis. Apremilast appears to be a potential new agent for the treatment of rheumatoid arthritis.