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Bone disease represents a major cause of morbidity and mortality in Multiple Myeloma (MM); primarily driven by osteoclasts whose differentiation is dependent on expression of RANKL by MM cells. Notably, costimulation by ITAM containing receptors (i.e., FcγR) can also play a crucial role in osteoclast differentiation. Modeling the pathology of the bone marrow microenvironment with an ex vivo culture system of primary human multiple myeloma cells, we herein demonstrate that FcγR-mediated signaling, via staphylococcal protein A (SpA) IgG immune-complexes, can act as a critical negative regulator of MM-driven osteoclast differentiation. Interrogation of the mode-of-action revealed that FcγR-mediated signaling causes epigenetic modulation of chromosomal 3D architecture at the RANK promoter; with altered spatial orientation of a proximal super enhancer. Combined this leads to substantial down-regulation of RANK at a transcript, protein, and functional level. These observations shed light on a novel mechanism regulating RANK expression and provide a rationale for targeting FcγR-signaling for the amelioration of osteolytic bone pathology in disease.

Psoriatic arthritis (PsA) is a heterogeneous disease involving multiple potential tissue domains. Most outcome measures used so far in randomized clinical trials do not sufficiently reflect this domain heterogeneity. The concept that pathogenetic mechanisms might vary across tissues within a single disease, underpinning such phenotype diversity, could explain tissue-distinct levels of response to different therapies. In this Review, we discuss the tissue, cellular and molecular mechanisms that drive clinical heterogeneity in PsA phenotypes, and detail existing tissue-based research, including data generated using sophisticated interrogative technologies with single-cell precision. Finally, we discuss how these elements support the need for tissue-based therapy in PsA in the context of existing and new therapeutic modes of action, and the implications for future PsA trial outcomes and design.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

Objectives Circulating myeloid precursors are responsible for post-natal osteoclast (OC) differentiation and skeletal health, although the exact human precursors have not been defined. Enhanced osteoclastogenesis contributes to joint destruction in rheumatoid arthritis (RA) and tumour necrosis factor (TNF) is a well-known pro-osteoclastogenic factor. Herein, we investigated the interplay between receptor activator of nuclear factor kappa-Β ligand (RANK-L), indispensable for fusion of myeloid precursors and the normal development of OCs, and TNF in directing the differentiation of diverse pre-OC populations derived from human peripheral blood. Methods Flow cytometric cell sorting and analysis was used to assess the potential of myeloid populations to differentiate into OCs. Transcriptomic, epigenetic analysis, receptor expression and inhibitor experiments were used to unravel RANK-L and TNF signalling hierarchy. Results TNF can act as a critical homoeostatic regulator of CD14+ monocyte (MO) differentiation into OCs by inhibiting osteoclastogenesis to favour macrophage development. In contrast, a distinct previously unidentified CD14−CD16−CD11c+ myeloid pre-OC population was exempt from this negative regulation. In healthy CD14+ MOs, TNF drove epigenetic modification of the RANK promoter via a TNFR1-IKKβ-dependent pathway and halted osteoclastogenesis. In a subset of patients with RA, CD14+ MOs exhibited an altered epigenetic state that resulted in dysregulated TNF-mediated OC homoeostasis. Conclusions These findings fundamentally re-define the relationship between RANK-L and TNF. Moreover, they have identified a novel pool of human circulating non-MO OC precursors that unlike MOs are epigenetically preconditioned to ignore TNF-mediated signalling. In RA, this epigenetic preconditioning occurs in the MO compartment providing a pathological consequence of failure of this pathway.

The γδ T cells reside predominantly at barrier sites and play essential roles in immune protection against infection and cancer. Despite recent advances in the development of γδ T cell immunotherapy, our understanding of the basic biology of these cells, including how their numbers are regulated in vivo, remains poor. This is particularly true for tissue-resident γδ T cells. We have identified the β(2) family of integrins as regulators of γδ T cells. β(2)-integrin–deficient mice displayed a striking increase in numbers of IL-17–producing Vγ6Vδ1(+) γδ T cells in the lungs, uterus, and circulation. Thymic development of this population was normal. However, single-cell RNA sequencing revealed the enrichment of genes associated with T cell survival and proliferation specifically in β(2)-integrin–deficient IL-17(+) cells compared to their wild-type counterparts. Indeed, β(2)-integrin–deficient Vγ6(+) cells from the lungs showed reduced apoptosis ex vivo, suggesting that increased survival contributes to the accumulation of these cells in β(2)-integrin–deficient tissues. Furthermore, our data revealed an unexpected role for β(2) integrins in promoting the thymic development of the IFNγ-producing CD27(+) Vγ4(+) γδ T cell subset. Together, our data reveal that β(2) integrins are important regulators of γδ T cell homeostasis, inhibiting the survival of IL-17–producing Vγ6Vδ1(+) cells and promoting the thymic development of the IFNγ-producing Vγ4(+) subset. Our study introduces unprecedented mechanisms of control for γδ T cell subsets.

Large-vessel vasculitis (LVV) manifests as inflammation of the aorta and its major branches and is the most common primary vasculitis in adults. LVV comprises two distinct conditions, giant cell arteritis and Takayasu arteritis, although the phenotypic spectrum of primary LVV is complex. Non-specific symptoms often predominate and so patients with LVV present to a range of health-care providers and settings. Rapid diagnosis, specialist referral and early treatment are key to good patient outcomes. Unfortunately, disease relapse remains common and chronic vascular complications are a source of considerable morbidity. Although accurate monitoring of disease activity is challenging, progress in vascular imaging techniques and the measurement of laboratory biomarkers may facilitate better matching of treatment intensity with disease activity. Further, advances in our understanding of disease pathophysiology have paved the way for novel biologic treatments that target important mediators of disease in both giant cell arteritis and Takayasu arteritis. This work has highlighted the substantial heterogeneity present within LVV and the importance of an individualized therapeutic approach. Future work will focus on understanding the mechanisms of persisting vascular inflammation, which will inform the development of increasingly sophisticated imaging technologies. Together, these will enable better disease prognostication, limit treatment-associated adverse effects, and facilitate targeted development and use of novel therapies.

ObjectivesMacrophage subsets, activated by T cells, are increasingly recognised to play a central role in rheumatoid arthritis (RA) pathogenesis. Janus kinase (JAK) inhibitors have proven beneficial clinical effects in RA. In this study, we investigated the effect of JAK inhibitors on the generation of cytokine-activated T (Tck) cells and the production of cytokines and chemokines induced by Tck cell/macrophage interactions.MethodsCD14+monocytes and CD4+T cells were purified from peripheral blood mononuclear cells from buffy coats of healthy donors. As representative JAK inhibitors, tofacitinib or ruxolitinib were added during Tck cell differentiation. Previously validated protocols were used to generate macrophages and Tck cells from monocytes and CD4+T cells, respectively. Cytokine and chemokine including TNF, IL-6, IL-15, IL-RA, IL-10, MIP1α, MIP1β and IP10 were measured by ELISA.ResultsJAK inhibitors prevented cytokine-induced maturation of Tck cells and decreased the production of proinflammatory cytokines TNF, IL-6, IL-15, IL-1RA and the chemokines IL-10, MIP1α, MIP1β, IP10 by Tck cell-activated macrophages in vitro (pConclusionsOur findings show that JAK inhibition disrupts T cell-induced macrophage activation and reduces downstream proinflammatory cytokine and chemokine responses, suggesting that suppressing the T cell-macrophage interaction contributes to the therapeutic effect of JAK inhibitors.

Annexin-A1 has a well-defined anti-inflammatory role in the innate immune system, but its function in adaptive immunity remains controversial. This glucocorticoid-induced protein has been implicated in a range of inflammatory conditions and cancers, as well as being found to be overexpressed on the T cells of patients with autoimmune disease. Moreover, the formyl peptide family of receptors, through which annexin-A1 primarily signals, has also been implicated in these diseases. In contrast, treatment with recombinant annexin-A1 peptides resulted in suppression of inflammatory processes in murine models of inflammation. This review will focus on what is currently known about annexin-A1 in health and disease and discuss the potential of this protein as a biomarker and therapeutic target.

Exercise is recommended as a non-pharmacological therapy for osteoarthritis (OA). Clinical studies investigating the impact of exercise on OA have primarily focussed on the assessment of joint pain and mobilisation, where positive outcomes have been demonstrated. Clinical imaging studies provide limited information on the impact of exercise (positive or negative) on the actual bone and soft tissue pathology. Various exercise regimes, with differing intensities and duration, have been used in a range of OA models, with disparate results. The present study provides definitive insight into the effect of moderate exercise on early joint pathology in the destabilisation of the medial meniscus (DMM) mouse model of OA. Exercise was induced by forced treadmill walking for 3 or 7 weeks. Joints were analysed by microcomputed tomography and histology. Exercise offered protection against cartilage damage and joint inflammation, and a temporary protection against osteosclerosis. Furthermore, exercise modified the metaphyseal trabecular microarchitecture of the osteoarthritic leg. Collectively, our findings provide scientific support for the clinical recommendation of moderate exercise as a physical therapy in OA. In addition to indirect benefit via positive physiological effects of weight loss, our data suggest direct short-term benefits in ameliorating pathology of cartilage, synovitis and bone.

Models of bone remodelling could be useful in drug discovery, particularly if the model is one that replicates bone regeneration with reduction in osteoclast activity. Here we use nanovibrational stimulation to achieve this in a 3D co-culture of primary human osteoprogenitor and osteoclast progenitor cells. We show that 1000 Hz frequency, 40 nm amplitude vibration reduces osteoclast formation and activity in human mononuclear CD14+ blood cells. Additionally, this nanoscale vibration both enhances osteogenesis and reduces osteoclastogenesis in a co-culture of primary human bone marrow stromal cells and bone marrow hematopoietic cells. Further, we use metabolomics to identify Akt (protein kinase C) as a potential mediator. Akt is known to be involved in bone differentiation via transforming growth factor beta 1 (TGFβ1) and bone morphogenetic protein 2 (BMP2) and it has been implicated in reduced osteoclast activity via Guanine nucleotide-binding protein subunit α13 (Gα13). With further validation, our nanovibrational bioreactor could be used to help provide humanised 3D models for drug screening.

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (PtenΔ/ΔBRF1Tg) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In PtenΔ/ΔBRF1Tg tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis.

Objectives Fatty acid oxidation (FAO) and glycolysis have been implicated in immune regulation and activation of macrophages. However, investigation of human monocyte intracellular metabolism in the context of the hypoxic and inflammatory rheumatoid arthritis (RA) synovium is lacking. We hypothesized that exposure of monocytes to the hypoxic and inflammatory RA environment would have a profound impact on their metabolic state, and potential to contribute to disease pathology. Methods Human monocytes were isolated from buffy coats and exposed to hypoxia. Metabolic profiling of monocytes was carried out by LC-MS metabolomics. Inflammatory mediator release after LPS or RA-synovial fluid (RA-SF) stimulation was analysed by ELISA. FAO was inhibited by etomoxir or enhanced with exogenous carnitine supplementation. Transcriptomics of RA blood monocytes and RA-SF macrophages was carried out by microarray. Results Hypoxia exacerbated monocyte-derived CCL20 and IL-1β release in response to LPS, and increased glycolytic intermediates at the expense of carnitines. Modulation of carnitine identified a novel role for FAO in the production of CCL20 in response to LPS. Transcriptional analysis of RA blood monocytes and RA-SF macrophages revealed that fatty acid metabolism was altered and CCL20 increased when monocytes enter the synovial environment. In vitro analysis of monocytes showed that RA-SF increases carnitine abundance and CCL20 production in hypoxia, which was exacerbated by exogenous carnitine. Conclusion This work has revealed a novel inflammatory mechanism in RA that links FAO to CCL20 production in human monocytes, which could subsequently contribute to RA disease pathogenesis by promoting the recruitment of Th17 cells and osteoclastogenesis.

Summary Psoriatic arthritis (PsA) is a heterogeneous inflammatory arthritis associated with psoriasis. Patients manifest variable presentations with potential involvement of peripheral joints, spine, tendons, skin, and nails. There has been a rapid expansion in targeted treatment options for patients with PsA, but typically less than half of those who receive therapy achieve optimal treatment targets. Many patients respond to second-line or third-line biological therapies, but little evidence exists to guide the choice of therapeutics for each individual. At present, choice of therapy is driven by active clinical disease domains, clinician familiarity with existing treatments, and cost. Here, we review recent data that highlight the potential for personalised, or precision, medicine in PsA and other forms of inflammatory arthritis, noting that this research is still at a preliminary stage. In the future, a combination of detailed immunophenotyping and sophisticated statistical analyses should help to facilitate a personalised medicine approach in PsA, following examples from other clinical areas, such as oncology. This change in approach to the treatment of PsA has the potential to maximise outcomes for patients and to provide optimal therapies without delay.

The alarmin S100A8/A9 is implicated in sterile inflammation-induced bone resorption and has been shown to increase the bone-resorptive capacity of mature osteoclasts. Here, we investigated the effects of S100A9 on osteoclast differentiation from human CD14

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

Objectives Psoriatic arthritis (PsA) is associated with bone erosion and inflammation-induced bone loss, which are mediated by osteoclasts (OC) and modulated by inflammatory cytokines. Apremilast (APR) (a selective phosphodiesterase 4 inhibitor) is efficacious in PsA and acts by inhibiting cytokine production. However, there are no direct data informing whether and how APR affects osteoclast formation in humans. Methods Osteoclastogenic cytokine production by activated human peripheral blood mononuclear cells (PBMCs) was measured in the presence and absence of APR. Effects of APR on osteoclast differentiation were tested (i) in co-cultures of activated PBMCs and human CD14+ blood monocytes as well as (ii) in CD14+ blood monocytes stimulated with activated-PBMCs supernatant, TNF or IL-17A. Bone resorption was measured on OsteoAssay plates. Effects of APR on ex vivo osteoclast differentiation were compared in PsA, pre-PsA and psoriasis patients, as well as in healthy controls. Results APR significantly impaired the expression of key osteoclastogenic cytokines in activated PBMCs. Furthermore, APR dose-dependently and significantly inhibited activated PBMC-driven osteoclast differentiation and ex vivo osteoclast differentiation of PBMCs derived from PsA and pre-PsA patients, but not from psoriasis patients or healthy controls. TNF and IL-17A-enhanced osteoclastogenesis and osteolytic activity of CD14+ blood monocytes from PsA patients was also significantly inhibited by APR. Finally, APR inhibited expression of the key osteoclast fusion protein dendritic cell-specific transmembrane protein. Conclusion Phosphodiesterase 4 targeting by APR not only inhibits osteoclastogenic cytokine production, but also directly suppresses inflammation-driven osteoclastogenesis. These data provide initial evidence that APR has the potential to provide a direct bone protective effect in PsA.

Ageing is a major risk factor for the development of cardiovascular disease (CVD) and cancer. Whilst the cumulative effect of exposure to conventional cardiovascular risk factors is important, recent evidence highlights clonal haematopoiesis of indeterminant potential (CHIP) as a further key risk factor. CHIP reflects the accumulation of somatic, potentially pro-leukaemic gene mutations within haematopoietic stem cells over time. The most common mutations associated with CHIP and CVD occur in genes that also play central roles in the regulation of inflammation. While CHIP carriers have a low risk of haematological malignant transformation (

Introduction and Objectives Severe coronavirus 19 disease (COVID 19) has rapidly emerged as a global health threat and, despite considerable advances, outcomes remain poor in many patients. Published data infers considerable heterogeneity, with 80% suffering minimal symptoms but a minority developing life-threatening disease. COVID 19 trials to-date have been necessarily broad but the emergence of established therapies (e.g. Dexamethasone) and distinct phenotypes (e.g. immune activated, prothrombotic) suggests that early stratification to licensed or trial agents might result in improved outcomes. The ASTERIX study aims to define disease endotypes, based on baseline biological signatures associated with COVID-19 pneumonia, development of respiratory failure and death, which could be targeted in future trials. Methods >6,000 samples of blood, urine and respiratory secretions were collected and banked during the first wave of the COVID 19 pandemic in Glasgow. The cohort is organised into Tiers 0, 1 & 2 with each tier having an increasing number of samples available for downstream translational research. All tiers have the same associated comprehensive clinical data including comorbidity, ethnicity, blood results, imaging, prescription data and outcomes, including critical care support and survival. Results Tier 0 contains 1,512 cases, Tier 1 (defined by having at least one surplus sample banked for downstream assays) contains ~1000 cases. Tier 2 (defined as having matched samples of serum, plasma and a buffy coat) contains 421 cases. Sample collation and data analysis is ongoing but preliminary review indicates a mortality rate of 29%, which is consistent with that reported in UK-wide COVID 19 series. The project team have made extensive links with collaborators and a scientific review board has been convened. The following projects are at various stages of approval and delivery: (1) Host Epigenomics (2) Host Proteomics (3) Host Metabolomics (4) miRNA Outcome Signatures (5) Host Respiratory Microbiome (6) COVID 19 Coagulopathy. Conclusions Data and banked samples will be used to develop endotypes (biological signatures derived from statistical models) associated with progression to key clinical outcomes. This information will be used to identify high-risk cohorts that could be targeted in future studies testing suitable interventions, as directed by the content of each signature.

Cigarette smoke stimulates an inflammatory response and produces oxidants that cause oxidative stress in the lung, promoting pathophysiological changes related to chronic obstructive pulmonary disease (COPD).1 Hydrogen peroxide (H2O2) is an important oxidant detected in breath condensate of COPD patients.2 We aim to understand how chronic exposure to H2O2 alone or in combination with other inflammatory mediators influences epithelial cell responses relevant to COPD lung pathology. BEAS-2B cells were exposed chronically to H2O2 for 2 h/day for 3 days at different concentrations, alone or in combination with TGF-β (10 ng/ml) or LPS (100 or 500 ng/ml). Cell viability was assessed by MTT assay. Cytokines were measured by ELISA. Intracellular ROS production was detected by CM-H2DCFDA assay. Data were analysed using one-way ANOVA, followed by Multiple Comparison Test. Cells tolerated a repeated exposure of H2O2 (up to 15 μM) ± TGF-β or LPS without significant loss of viability. Intracellular ROS was significantly elevated in the presence of LPS (mean ± SEM; 217±17%; p Oxidative stress appears to be generated in BEAS-2B cells by LPS or H2O2 alone, and increased in combination. Repeated exposure to H2O2 induced minimal inflammatory response, but synergistically enhanced the effect of TGF-β and LPS on cytokine production. These data suggest combined exposure models may be useful to study the effects of epithelial cell challenges relevant to COPD pathology. References Kirkham P, Rahman I. Oxidative stress in asthma and COPD: antioxidant as a therapeutic strategy. Pharmacol Ther 2006;111:476–94. Montuschi, P. Exhaled breath condensate analysis in patients with COPD. Clin Chim Acta 2005;356:22–34.

The NF-ĸB transcription factor is a critical regulator of immune homeostasis and inflammatory responses and is a critical factor in the pathogenesis of inflammatory disease. The pathways to NF-ĸB activation are paradigms for signal-induced ubiquitination and proteasomal degradation, control of transcription factor function by subcellular localisation, and the control of gene transcription and physiological processes by signal transduction mechanisms. Despite the importance of NF-ĸB in disease, the NF-ĸB pathway remains unexploited for the treatment of inflammatory disease. Our understanding of NF-ĸB comes mostly from studies of transgenic mice and cell lines where components of the pathway have been deleted or over expressed. Recent advances in quantitative proteomics offer new opportunities to understand the NF-ĸB pathway using the absolute abundance of individual pathway components. We have analysed available quantitative proteomic datasets to establish the structure of the NF-ĸB pathway in human immune cells under both steady state and activated conditions. This reveals a conserved NF-κB pathway structure across different immune cell lineages and identifies important differences to the current model of the NF-ĸB pathway. These include the findings that the IKK complex in most cells is likely to consist predominantly of IKKβ homodimers, that the relative abundancies of IκB proteins show strong cell type variation, and that the components of the non-canonical NF-ĸB pathway are significantly increased in activated immune cells. These findings challenge aspects of our current view of the NF-κB pathway and identify outstanding questions important for defining the role of key components in regulating inflammation and immunity., Highlights • Quantitative proteomic datasets offer new insights into the NF-κB pathway. • The structure of the NF-κB pathway is highly conserved in human immune cells. • The IKK complex is likely composed mainly of IKKβ homodimers. • The relative abundancies of IκBα, −β and -ε show strong cell type variation. • Components of the non-canonical NF-ĸB pathway are greatly increased by activation.

Background Pelvic nodal metastasis in prostate cancer impacts patient outcome negatively. Objective To explore tumor-infiltrating immune cells as a potential predictive tool for regional lymph node (LN) metastasis. Design, setting, and participants We applied multiplex immunofluorescence and targeted transcriptomic analysis on 94 radical prostatectomy specimens in patients with (LN+) or without (LN–) pelvic nodal metastases. Both intraepithelial and stromal infiltrations of immune cells and differentially expressed genes (mRNA and protein levels) were correlated with the nodal status. Outcome measurements and statistical analysis The identified CD4 effector cell signature of nodal metastasis was validated in a comparable independent patient cohort of 184 informative cases. Patient outcome analysis and decision curve analysis were performed with the CD4 effector cell density–based signature. Results and limitations In the discovery cohort, both tumor epithelium and stroma from patients with nodal metastasis had significantly lower infiltration of multiple immune cell types, with stromal CD4 effector cells highlighted as the top candidate marker. Targeted gene expression analysis and confirmatory protein analysis revealed key alteration of extracellular matrix components in tumors with nodal metastasis. Of note, stromal CD4 immune cell density was a significant independent predictor of LN metastasis (odds ratio [OR] = 0.15, p = 0.004), and was further validated as a significant predictor of nodal metastasis in the validation cohort (OR = 0.26, p, Take Home Message Decreased intratumoral CD4 T effector cell densities are associated with a higher risk of lymph node metastasis. Future evaluation of CD4-based assays on prostate cancer diagnostic biopsy materials may improve selection of at-risk patients for the treatment of lymph node metastasis.

Background Our knowledge of immune-mediated inflammatory disease (IMID) aetiology and pathogenesis has improved greatly over recent years, however, very little is known of the factors that trigger disease relapses (flares), converting diseases from inactive to active states. Focussing on rheumatoid arthritis (RA), the challenge that we will address is why IMIDs remit and relapse. Extrapolating from pathogenetic factors involved in disease initiation, new episodes of inflammation could be triggered by recurrent systemic immune dysregulation or locally by factors within the joint, either of which could be endorsed by overarching epigenetic factors or changes in systemic or localised metabolism. Methods The BIO-FLARE study is a non-randomised longitudinal cohort study that aims to enrol 150 patients with RA in remission on a stable dose of non-biologic disease-modifying anti-rheumatic drugs (DMARDs), who consent to discontinue treatment. Participants stop their DMARDs at time 0 and are offered an optional ultrasound-guided synovial biopsy. They are studied intensively, with blood sampling and clinical evaluation at weeks 0, 2, 5, 8, 12 and 24. It is anticipated that 50% of participants will have a disease flare, whilst 50% remain in drug-free remission for the study duration (24 weeks). Flaring participants undergo an ultrasound-guided synovial biopsy before reinstatement of previous treatment. Blood samples will be used to investigate immune cell subsets, their activation status and their cytokine profile, autoantibody profiles and epigenetic profiles. Synovial biopsies will be examined to profile cell lineages and subtypes present at flare. Blood, urine and synovium will be examined to determine metabolic profiles. Taking into account all generated data, multivariate statistical techniques will be employed to develop a model to predict impending flare in RA, highlighting therapeutic pathways and informative biomarkers. Despite initial recruitment to time and target, the SARS-CoV-2 pandemic has impacted significantly, and a decision was taken to close recruitment at 118 participants with complete data. Discussion This study aims to investigate the pathogenesis of flare in rheumatoid arthritis, which is a significant knowledge gap in our understanding, addressing a major unmet patient need. Trial registration The study was retrospectively registered on 27/06/2019 in the ISRCTN registry 16371380.

Mucosal immunology 14(3), 717-727 (2021). doi:10.1038/s41385-020-00366-3, Published by Nature Publishing Group, New York, NY

The immune system plays a crucial role in maintaining a healthy body by working around the clock to recognize and respond to infection. Inflammation is part of the immune system’s protective response to an infection. The inflammatory response is incredibly powerful, so much so that it can damage the body’s cells if it is not tightly controlled. Sometimes, inflammation affects the whole body—this is called sepsis. The powerful and complex mechanisms in place to wipe out the infection can cause serious damage to healthy cells and tissues. Uncontrolled inflammation can cause irreversible damage to the body’s organs, such as the kidneys, eventually causing organs to shut down. If sepsis is not treated rapidly, it can lead to death. In this article, we describe the symptoms and diagnosis of sepsis and some of the current research being performed to better understand this dangerous process.

SARS-COV-2 vaccines have been shown to be efficacious primarily in healthy volunteer populations and population level studies. Immune responses following SARS-CoV-2 vaccination are less well characterised in potentially immune vulnerable patient groups, including those with immune-mediated inflammatory and chronic diseases (inflammatory arthritis [IA] incorporating rheumatoid arthritis [RA] and psoriatic arthritis [PsA]; ANCA-Associated Vasculitis [AAV]; inflammatory bowel disease [IBD]); hepatic disease (HepD), end stage kidney disease requiring haemodialysis (HD) without or with immunosuppression (HDIS); solid cancers (SC) and haematological malignancies (HM), and those that have undergone haemopoietic stem cell transplant (HSCT). The OCTAVE trial is a multi-centre, multi-disease, prospective cohort that will comprehensively assess SARS-CoV-2 vaccine responses within and between the abovementioned disease cohorts using common analytical platforms in patients recruited across the United Kingdom (UK). The majority of subjects received either COVID-19 mRNA Vaccine BNT162b2 (Pfizer/BioNTech) or ChAdOx1 Vaccine (AstraZeneca formerly AZD1222) as part of the UK National COVID19 vaccination programme. As of 13 th August 2021; 2,583 patients have been recruited. We report herein the humoral and T cell immune response results from the first 600 participants recruited where serology data are available at baseline, pre-second vaccine dose (boost) and/or 4 weeks post second dose. We also include in the analysis, data obtained from 231 healthy individuals from the PITCH (Protective Immunity from T cells in Healthcare workers) study. Overall, in comparison to PITCH where 100% of tested individuals (n=93) generated anti-Spike antibodies after vaccine doses, 89% of patients within OCTAVE seroconverted 4 weeks after second vaccine dose. By corollary, approximately 11% of patients across all disease cohorts fail to generate antibodies that react to SARS-CoV-2 spike 4 weeks after two vaccines. Failure to generate spike reactive antibodies was found at a higher proportion in some specific patient subgroups, particularly AAV (72.4%), HD-IS (16.7%) and HepD (16.7%). Importantly, all recruited AAV patients had received Rituximab; a targeted B cell depletion therapy. Furthermore, even in those who seroconverted, 40% of patients across disease cohorts generate lower levels of SARS-CoV-2 antibody reactivity compared to healthy subjects after two SARS-CoV-2 vaccines; the functional significance of these findings in providing protection from subsequent SARS-CoV-2 exposure is not currently known. In contrast to the observed serological response, evaluation of the Spike-specific T cell response revealed that across all patient sub-groups (including AAV) a response similar to healthy individuals was generated. Our data argue strongly for further vaccination strategies to optimise humoral immune responses against SARS-CoV-2 in patients with chronic diseases and/or patients on immune suppressive therapies. Trial Registration: The trial is registered on ISRCTN 12821688. Funding: This work was supported by the Medical Research Council COVID-19 Immunity – National Core Study (IMM-NCS) [grant number MC-PC-20031]. Staff at the Cancer Research UK Clinical Trials Unit (CRCTU) are supported by a core funding grant from Cancer Research UK (C22436/A25354). PK and EB are supported by the NIHR Birmingham Biomedical Research Centres at the University Hospitals Birmingham NHS Foundation Trust and the University of Birmingham Biomedical Research Centres. EB and PK are supported by an NIHR Senior Investigator award. PK is funded by WT109965MA. SJD is funded by an NIHR Global Research Professorship (NIHR300791). TdS is funded by a Wellcome Trust Intermediate Clinical Fellowship (110058/Z/15/Z). DS is supported by the NIHR Academic Clinical Lecturer programme in Oxford. LT is supported by the Wellcome Trust (grant number 205228/Z/16/Z), the U.S. Food and Drug Administration Medical Countermeasures Initiative contract 75F40120C00085. and the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections (NIHR200907) at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford. The PITCH (Protective Immunity from T cells to Covid-19 in Health workers) Consortium, is funded by the UK Department of Health and Social Care with contributions from UKRI/NIHR through the UK Coronavirus Immunology Consortium (UKCIC), the Huo Family Foundation and The National Institute for Health Research (UKRIDHSC COVID-19 Rapid Response Rolling Call, Grant Reference Number COV19-RECPLAS). Declaration of Interest: None to declare. Ethical Approval: This study was approved by the UK Medicines and Healthcare Products Regulatory Agency on the 5th February 2021 and the London and Chelsea Research Ethics Committee (REC Ref:21/HRA/0489) on 12th February 2021, with subsequent amendments approved on 3rd March 2021, 19th April 2021 and 26th April 2021).

Background Once bulk RNA-seq data has been processed, i.e. aligned and then expression and differential tables generated, there remains the essential process where the biology is explored, visualized and interpreted. Without the use of a visualisation and interpretation pipeline this step can be time consuming and laborious, and is often completed using R. Though commercial visualisation and interpretation pipelines are comprehensive, freely available pipelines are currently more limited. Results Here we demonstrate Searchlight, a freely available bulk RNA-seq visualisation and interpretation pipeline. Searchlight provides: a comprehensive statistical and visual analysis, focusing on the global, pathway and single gene levels; compatibility with most differential experimental designs irrespective of organism or experimental complexity, via three workflows; reports; and support for downstream user modification of plots via user-friendly R-scripts and a Shiny app. We show that Searchlight offers greater automation than current best tools (VIPER and BioJupies). We demonstrate in a timed re-analysis study, that alongside a standard bulk RNA-seq processing pipeline, Searchlight can be used to complete bulk RNA-seq projects up to the point of manuscript quality figures, in under 3 h. Conclusions Compared to a manual R based analysis or current best freely available pipelines (VIPER and BioJupies), Searchlight can reduce the time and effort needed to complete bulk RNA-seq projects to manuscript level. Searchlight is suitable for bioinformaticians, service providers and bench scientists. https://github.com/Searchlight2/Searchlight2.

Objective: \ud We undertook this study to examine microRNA (miRNA) expression across rheumatoid arthritis (RA) phenotypes, along with the effects and mechanisms of action of miRNA‐17‐5p (miR‐17).\ud \ud Methods: \ud A miRNA array was performed in synovial tissue biopsied from patients with naive erosive RA (n = 3) and patients with nonerosive RA (n = 3). MicroRNA‐17 lipoplex was delivered intraarticularly in the murine collagen‐induced arthritis model. Clinical, histologic, and structural effects were studied over the course of arthritis. In‐depth studies of the mechanisms of action of miR‐17 were performed in primary RA fibroblast‐like synoviocytes (FLS) isolated from synovial tissue.\ud \ud Results: \ud Fifty‐five miRNAs including miR‐17 were reduced in erosive RA. The miR‐17 transfection into arthritic paws reduced the clinical inflammation score between day 2 and day 7 (2.8 versus 1.9; P = 0.03). Synovial B cell, T cell, macrophage, and polynuclear neutrophil infiltration was significantly reduced. Structural damage was also decreased, as shown by a reduction in the number of osteoclasts detected using tartrate‐resistant acid phosphatase staining (osteoclast surface/bone surface 32% versus 18%; P = 0.005) and erosion score by computed tomography analysis (2.9 versus 1.7; P = 0.023). Proinflammatory cytokines from the interleukin‐6 (IL‐6) family and IL‐1β expression were also significantly reduced, but tumor necrosis factor was not. MicroRNA‐17 directly targeted the 3′‐untranslated regions of STAT3 and JAK1. STAT3 and JAK1 messenger RNA (mRNA) and protein expression were reduced in RA FLS following miR‐17 transfection. STAT3 and JAK1 mRNA and activation of STAT3, as assessed by immunohistochemistry, were also reduced in injected paws (% stained area 93% versus 62%; P = 0.035).\ud \ud Conclusion: \ud We demonstrate an antiinflammatory and antierosive role of miR‐17 in vivo. This effect involves the suppression of the IL‐6 family autocrine‐amplifying loop through the direct targeting of JAK1 and STAT3.

Changes in the plasma metabolic profile were characterised in newly diagnosed rheumatoid arthritis (RA) patients upon commencement of conventional disease-modifying anti-rheumatic drug (cDMARD) therapy. Plasma samples collected in an early RA randomised strategy study (NCT00920478) that compared clinical (DAS) disease activity assessment with musculoskeletal ultrasound assessment (MSUS) to drive treatment decisions were subjected to untargeted metabolomic analysis. Metabolic profiles were collected at pre- and three months post-commencement of nonbiologic cDMARD. Metabolites that changed in association with changes in the DAS44 score were identified at the three-month timepoint. A total of nine metabolites exhibited a clear correlation with a reduction in DAS44 score following cDMARD commencement, particularly itaconate, its derived anhydride and a derivative of itaconate CoA. Increasing itaconate correlated with improved DAS44 score and decreasing levels of C-reactive protein (CRP). cDMARD treatment effects invoke consistent changes in plasma detectable metabolites, that in turn implicate clinical disease activity with macrophages. Such changes inform RA pathogenesis and reveal for the first time a link between itaconate production and resolution of inflammatory disease in humans. Quantitative metabolic biomarker-based tests of clinical change in state are feasible and should be developed around the itaconate pathway.

Background Septic arthritis (SA) caused by bacterial species, such as Staphylococcus aureus, has high morbidity and mortality. Currently diagnosis is often prolonged and unreliable, with no suitable near-patient biomarkers available. To generate more reliable biomarkers and to understand pathogenesis we sought to develop a novel ex vivo system to explore the effect of pathogenic Staph. aureus strains in promoting cartilage degradation. Methods Human cartilage explants were obtained from femoral heads being surgically removed following trauma. Explants were infected for 48h with 106 cfu bacteria from two Staph. aureus SA-derived patient isolates (28g & 36v strains). In the final 24h of bacterial infection, neutrophils purified from healthy donor blood were added to explant cultures at 3 x 106 cells/well. Chondrocyte viability was assessed using CellTracker green CMFDA and propidium iodide. Images were captured using confocal microscopy (LSM880) and cells counted using Imaris software. Structural damage was measured by glycosaminoglycan (GAG) and a neo-epitope of MMP-mediated degradation of type II collagen (C2M) release. Statistical analysis was performed using GraphPad Prism software. Results When cartilage explants were co-cultured with bacteria +/- neutrophils, cell death was significantly increased compared to the negative control or addition of neutrophils alone, (Friedman multiple comparisons test, N = 3, negative control vs. bacteria - neutrophils p Conclusion A co-culture model of septic arthritis has been developed which allows precise examination of the contribution of the host neutrophil response to cartilage damage. We used this to identify a collagen breakdown product as a biomarker of host response to infected cartilage. This novel model will be a valuable tool in understanding the pathology of joint infection and can be used for the identification of future diagnostic biomarkers. Disclosures K.E. McCall None. C. Atherton None. C. Thudium None. C. Goodyear Grants/research support; C.G. has received funding for research from Celgene, AstraZeneca, MedAnnex, UCB & Jannsen. T. Evans None. N. Millar Grants/research support; Novartis. I. McInnes Consultancies; I.M. has received consultancies fees from BMS, Abbvie, Lilly, GSK & Pfizer. Grants/research support; I.M received research funding from Calgene, Janssen, Novartis, Boehringer Ingelheim & BMS.

Angiotensin II-type 1 receptor stimulation is recognised to promote inflammation, a state central to the development and maintenance of rheumatoid arthritis. Herein we examined the use of losartan, an angiotensin II-type 1 receptor antagonist, on vascular reactivity, knee joint diameter and behavioural assessment of pain in a Freund's complete adjuvant (FCA) mouse model of joint inflammation. Monoarthritis was induced via FCA in the presence or absence of losartan with naive mice serving as controls. Knee joint swelling, joint pain (assessed by dynamic weight bearing of limb use), knee joint artery reactivity (assessed ex vivo) and blood perfusion of the knee joint (assessed in vivo) were determined. FCA mediated a significant increase in knee joint diameter and reduced weight-bearing (a surrogate for pain sensation) of the affected limb. Notably, these phenomena were substantially reduced when mice were prophylactically treated with losartan. Assessment of arterial relaxation and blood perfusion with acetylcholine stimulation revealed that FCA resulted in significant vascular dysfunction, which was resolved to naïve levels with losartan treatment. Through the actions of losartan, these findings indicate that the angiotensin II-type 1 receptor is a likely therapeutic target of importance in the development of the physical changes, pain sensation and vascular dysfunction found in inflammatory arthritis.

Objective To investigate the relationship between intestinal inflammation and the central and peripheral innate immune system, in the pathogenesis of HLA-B27 associated spondyloarthritis. Methods The myeloid compartment of the bone marrow and blood of HLA-B27 transgenic (B27), control HLA-B7 transgenic (B7), and non-transgenic rats were evaluated by flow cytometry. Plasma from rats were assessed by ELISA for CCL2 and IL-1α levels. Rats were treated for 4 weeks with antibiotics and the blood and bone marrow myeloid compartments were evaluated by flow cytometry. The osteoclastogenic potential of bone marrow cells from antibiotic treated rats, in the presence or absence of TNFα, was evaluated in vitro. Results B27 rats have substantially higher numbers of circulating Lin-CD172a+CD43l° monocytes than control animals, which significantly correlates with higher levels of plasma CCL2. Antibiotic treatment of B27 rats markedly reduced ileitis, plasma CCL2 and IL-1α levels, and the number of bone marrow and blood Lin-CD172a+CD43l° monocytes, which have the greatest in vitro osteoclastogenic potential. Antibiotic treatment also prevented the TNFα-dependent enhancement of osteoclastogenesis in transgenic B27 rats. Conclusions The microbiota-dependent intestinal inflammation in B27 rats directly drives the systemic inflammatory and bone erosive potential of the monocyte compartment. This article is protected by copyright. All rights reserved.

Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis. Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14+ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7 days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted. EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n = 11) and RA-derived (n = 12) EXOs profoundly inhibited osteoclast differentiation (70%, p

Inflammatory cytokine production is a hallmark of COPD. PAR2 activation, via the transmembrane serine protease matriptase, results in the regulation of pro-inflammatory cytokines, including IL-6 and IL-8 (Seitz et al., 2007). The aim of this study was to investigate a putative role for PAR2 in COPD. PAR2 and matriptase expression was determined by immunofluorescence in primary human bronchial epithelial cells derived from healthy controls and COPD patients (HBECs & DHBECs respectively). Levels of secreted IL-6 and IL-8 were evaluated by ELISA. The role of PAR2 in the DHBEC-associated inflammatory response was investigated using the PAR2 antagonist AZ8838 (Cheng et al., 2017). Immunofluorescent microscopy showed both HBECs and DHBECs express PAR2, whereas only DHBECs express matriptase. Evaluation of spontaneous cytokine secretion revealed that both IL-6 and IL-8 were significantly increased (P This study used a recently developed antagonist to demonstrate a role for PAR2 in the regulation of pro-inflammatory cytokine release from COPD bronchial epithelial cells. Since increased protease activity is a feature of COPD, elevated expression of matriptase may contribute to PAR2 activation in this disease. References Cheng, R. K. Y. et al. ( 2017) ‘Structural insight into allosteric modulation of protease-activated receptor 2’, Nature. NaturePublishing Group, 545(7652), pp. 112–115. doi: 10.1038/nature22309. Seitz, I. et al. ( 2007) ‘Membrane-type serine protease-1/matriptase induces interleukin-6 and -8 in endothelial cells by activation of protease-activated receptor-2: Potential implications in atherosclerosis’, Arteriosclerosis, Thrombosis, and Vascular Biology. doi: 10.1161/01.ATV.0000258862.61067.14.

Osteoarthritis (OA) is the most prevalent of the musculoskeletal conditions and represents a significant public health burden. While degeneration of articular cartilage is a key feature, it is now increasingly recognized as a complex condition affecting the whole joint, with synovial inflammation present in a significant proportion of patients. As a secretory tissue, the OA synovium is a rich source of both soluble inflammatory mediators and extracellular vesicles, including exosomes, which have been implicated in cell–cell communication. Exosome cargo has been found to include proteins, lipids and various RNA subtypes such as mRNA and miRNA, potentially capable of regulating gene expression in target cells and tissues. Profiling of exosome cargo and understanding effects on cartilage could elucidate novel regulatory mechanisms within the joint, providing insight for targeted treatment. The aim of this article is to review current literature on exosome biology, highlighting the relevance and application for OA pathogenesis.

Background Patients suffering from Psoriatic arthritis (PsA) commonly develop bone erosions and inflammatory-induced bone loss. This process is mediated by osteoclasts derived from monocytic precursors, and modulated by inflammatory cytokines (i.e. TNF, IL-1, IL-6, IL-17, IL-10 and GM-CSF) from immune and stromal cells. In immune cells (including CD14+ osteoclast pre-cursors), PDE4, an enzyme responsible for hydrolysing cyclic AMP to inactive AMP, drives inflammatory effects [1]. Importantly, Apremilast (APR; a selective PDE4 inhibitor) has known efficacy in PsA [2], and decreases pro-inflammatory mediators whilst increasing anti-inflammatory mediators (IL-10) [3]. Although published data indirectly suggest a positive impact of APR on bone in PsA, data is lacking with regard to the impact on bone resorption. Objectives To evaluate the impact of a selective inhibition of PDE4 by APR on osteoclastogenesis from human CD14+ precursors. Methods Osteoclasts were differentiated from primary human CD14+ blood monocytes (healthy controls) with RANKL and MCSF, in the presence or absence of APR. To specifically study the impact of APR on osteoclastogenesis in an inflammatory context, osteoclastogenesis was also undertaken in the presence of: (i) TNF, (ii) Supernatants from activated Peripheral Blood Mononuclear Cells (PBMC) or activated CD3 cells, treated with or without APR, (iii) Co-culture with activated PBMC or activated CD3 cells, treated with or without APR. TRAP+ multinucleated cells (mature osteoclasts) were enumerated via microscopy. Results In a non-inflammatory context, PDE4 inhibition by APR did not affect the differentiation of CD14+ precursors into mature osteoclasts. However, TNF-enhanced osteoclastogenesis was significantly decreased by APR (-30.0% +/- 14.9; p=0.0279).The treatment of either activated PBMCs or purified CD3+ T cells with APR substantially reduced cellular activation. In PBMCs this decrease in cellular activation resulted in a decrease in conditioned media-driven osteoclastogenesis (-49.7% +/- 13.2; p=0.0385). In comparison, APR treatment of purified CD3+ T cells did not reduce their osteoclastogenic potential. Conclusion The results of this study reveal that PDE4 targeting potently inhibits inflammatory-driven osteoclastogenesis. Moreover, these data also suggest that CD3+ T cells are not the main target of PDE4 inhibition in this context. In summation, our study supports the hypothesis that APR can modulate bone integrity in inflammatory condition such as PsA. References [1] Houslay. Drug Discov Today. 2005Nov15;10(22):1503-19. [2] Edwards. Ann Rheum Dis. 2016Jun;75(6):1065-73. Doi: 10.1136/annrheumdis-2015-207963. [3] Schafer. J Immunol Res. 2015;2015:906349. Doi: 10.1155/2015/906349. Acknowledgement YD received a fellowship from the Societe Francaise de Rhumatologie Disclosure of Interests Yannick Degboe Grant/research support from: Celgene PARTNER Fellowship, Iain McInnes Grant/research support from: AstraZeneca, Celgene, Compugen, Novartis, Roche, UCB Pharma, Consultant for: AbbVie, Celgene, Galvani, Lilly, Novartis, Pfizer, UCB Pharma, Carl Goodyear Grant/research support from: AstraZeneca, BMS, Celgene, Janssen, MedAnnex, Pfizer and UCB, Speakers bureau: Abbvie

Background Musculoskeletal conditions (MSC), such as Rheumatoid arthritis (RA), place a heavy burden on society and have severe consequences for the individuals affected. In order to limit this burden of disease, early diagnosis and implementation of treatment are essential and result in a significantly increased chance of achieving long-term sustained remission. The European League Against Rheumatism (EULAR) campaign ‘Don’t Delay Connect Today’ (DDCT) was created in order to educate the general public and primary healthcare providers (such as General Practitioners) about the importance of recognising the early warning signs of MSC’s. Many countries have adopted the campaign across Europe, with groups using new and creative ways to engage local communities with this essential message. In Scotland, the Rheumatosphere team have worked to develop interesting and impactful ways of disseminating this message to the public. Augmented reality (AR) is a potential tool to enhance learning and has already been successfully used as a new and exciting tool for teaching1. However, its role in less formal educational setting, such as public engagement, is still relatively unknown. Objectives The primary objective of this study was to test a new AR modality, assessing its effectiveness in increasing knowledge pertaining to RA and the central message of the DDCT campaign. Methods An interactive AR application was designed for a lay audience, incorporating aspects of RA disease pathogenesis along with the importance of early diagnosis and treatment of disease. The modality consisted of printed posters that were enhanced by an interactive AR application accessed through a hand-held tablet device. Members of the public, visiting the Glasgow Science Centre, were asked to assess our AR application by completing a 5 point Likert scale questionnaire before and after interacting with our posters and AR application. Results In total 27 participants took part in the testing, with the majority being between the age of 25-34 years old, a key target audience for the campaign, as this demographic commonly believe that they are ‘too young’ to develop arthritis2. Overall the testing revealed that the AR application was easy to use, engaging and enjoyable. Further evaluation conducted using a 5-point Likert scale, showed that the AR application was successful in raising awareness of RA, with 81% of the participants reporting that they felt more aware about the pathogenesis, symptoms and treatment of RA after use. Moreover, 55% of the participants thought that they would inform friends and family about the causes, symptoms and treatments of RA, helping to disseminate the campaign message further, enhancing it’s overall reach from a single event. Conclusion Overall the application was well received and indicates that this tool could be used to enhance public engagement moving forward. It would therefore be worthwhile to invest in the development of similar modalities for the EULAR ‘Don’t Delay, Connect Today’ campaign. References [1] Sommerauer, P. and Muller, O. (2014) ‘Augmented reality in informal learning environments: A field experiment in a mathematics exhibition’, Computers and Education. Elsevier Ltd, 79(2014), pp. 59–68. doi: 10.1016/j.compedu.2014.07.013. [2] Doran MF, Pond GR, Crowson CS, O’Fallon WM, Gabriel SE. Trends in incidence and mortality in rheumatoid arthritis in Rochester, Minnesota, over a forty-year period. Arthritis Rheum 2002;46:625-31.3. Disclosure of Interests Timea Kosa: None declared, Daniel Livingstone: None declared, Brian Loranger: None declared, Carl Goodyear Grant/research support from: AstraZeneca, BMS, Celgene, Janssen, MedAnnex, Pfizer and UCB, Speakers bureau: Abbvie, Louise Bennett : None declared

Background: Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with substantial local and systemic bone loss. Despite the availability of several treatment options many patients do not reach low disease activity. Furthermore, current therapeutics generally target inflammation rather than erosive pathology. Thus, there remains a need for new therapies that can target both aspects of the disease. Prior studies have shown that biphenylcarboxylic acid small molecule derivatives not only inhibit murine osteoclastogenesis but also attenuate inflammation and bone destruction in murine models of RA1,2. Objectives: To evaluate a novel small molecule derivative, MBS2133, on human osteoblastogenesis, osteoclastogenesis and cellular function, and to investigate the in vitro mechanism-of-action. Methods: Osteoblasts were derived from human mesenchymal stem cells. Cells were differentiated in the presence or absence of MBS2133 and mineralization assessed by Alizarin Red staining. Human CD14+ blood monocytes were differentiated into osteoclasts (OCs) with M-CSF and RANK-L, in the presence or absence of MBS2133, and/or metabolites. Mature OCs were stained with tartrate-resistant acid phosphatase (TRAP) and quantified by light microscopy. Osteolytic activity was assessed on mineral-coated surfaces. Western blot analysis was used to assess downstream signalling pathways. Changes in the metabolic profile of pre-osteoclasts following 4h exposure to MBS2133 was carried out by liquid chromatography mass spectrometry. Results: MBS2133 had no effect on the differentiation and function of primary human osteoblasts. In comparison, exposure of RANK-L stimulated CD14+ monocytes to MBS2133 significantly reduced OC differentiation and osteolytic activity of mature OCs. Notably, exposure of pre-OCs to MBS2133 for 2h at initiation of osteoclastogenesis, was sufficient to significantly reduce subsequent OC differentiation. Evaluation of treated pre-osteoclasts revealed that RANKL-mediated phosphorylation of p38 was reduced. Metabolomic analysis of pre-osteoclasts revealed that MBS2133 induced a substantial reduction in a range of metabolites associated with glycolysis, oxidative phosphorylation and fatty acid oxidation pathway. Notably, L-carnitine, which facilitates the transportation of fatty acids to the mitochondrial matrix and enables processing and entry into tricarboxylic acid (TCA) cycle for further energy production, was significantly reduced. In vitro supplementation of L-carnitine inhibited the ability of the compound to switch off OC differentiation and osteolytic activity. Conclusion: The results of this study demonstrate that MBS2133 specifically modulates the metabolome of myeloid cells, which has a substantial impact on their ability to differentiate into mature osteoclasts. These findings highlight the importance of modulating the glycolysis/oxidative phosphorylation axis in osteoclastogenesis and suggest that targeting the metabolic state of pre-osteoclasts could offer a new therapeutic approach to treat bone resorption in rheumatic diseases. References [1] Greig IR, et al. Development and Characterization of Biphenylsulfonamides as Novel Inhibitors of Bone Resorption. J.Med.Chem. 2006;49:7487–7492 [2] Coste E, et al. Identification of small molecule inhibitors of RANKL and TNF signalling as anti-inflammatory and antiresorptive agents in mice. AnnRheumDis2015;74:220–6. Disclosure of Interests: Shatakshi Sood: None declared, Lisa Patel Shareholder of: Shareholder of Istesso Ltd, Employee of: Employee of Istesso, Martyn Foster Shareholder of: AstraZeneca, Consultant for: Istesso, Levicept, Employee of: AstraZeneca, Louise Jopling Shareholder of: Johnson and Johnson (employee), Employee of: Employee of Janssen (Pharmaceutical arm of Johnson & Johnson) since May 2008 to present day, Rob van’t Hof Shareholder of: OsteoRx Ltd, Iain Greig Shareholder of: Shareholder in OsteoRx Ltd, a spin-out company from the University of Aberdeen, which retains a financial interest in MBS2133, Sam Williams Shareholder of: Shareholders of Istesso Ltd, Employee of: Employees of Istesso, Iain McInnes Grant/research support from: AstraZeneca, Celgene, Compugen, Novartis, Roche, UCB Pharma, Consultant for: AbbVie, Celgene, Galvani, Lilly, Novartis, Pfizer, UCB Pharma, Carl Goodyear Grant/research support from: AstraZeneca, BMS, Celgene, Janssen, MedAnnex, Pfizer and UCB, Speakers bureau: Abbvie

Background High levels of the damage-associated molecular pattern (DAMP) S100A8/A9 are produced in the inflamed synovium during experimental and human rheumatoid arthritis (RA), which have been implicated in sterile inflammation-induced bone resorption. We and others have previously shown that stimulation of mature osteoclasts with S100A8/A9 increases their bone-resorptive capacity. In agreement, reduced bone destruction was observed after induction of experimental RA models in S100a9-/- mice. However, its effects on the differentiation of osteoclasts from their precursors remains elusive. Objectives Here, we investigated the effects of S100A9 on osteoclast differentiation from CD14+ circulating precursors. Methods CD14+ monocytes were isolated from buffy coats of healthy donors and differentiated towards osteoclasts with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B (RANK) ligand in the presence or absence of S100A9. Differentiation state of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP) staining and resorption capacity was quantified using hydroxyapatite-like-coated plates. RNA expression was analyzed with RNA sequencing and qPCR. RANK expression was assessed using FACS. Underlying epigenetic programming was studied using chromatin immunoprecipitation. Secretion of pro-/anti-inflammatory mediators was analyzed with Luminex analysis. Results S100A9 stimulation during monocyte-to-osteoclast differentiation resulted in a strong decrease in the numbers of multinucleated osteoclasts, underlined by a decreased resorptive capacity. The thus differentiated cells showed a high production of pro-inflammatory factors, such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNFα) after 16h of stimulation. In contrast, at day 4, the cells showed a decreased expression of the osteoclast-promoting factor TNFα. Interestingly, we showed that S100A9 stimulation only during the first 16h of culture was sufficient to reduce osteoclastogenesis. To determine the mechanism of how this short S100A9 stimulation might reduce the osteoclast differentiation, we determined the protein expression of RANK. We observed that within this 16h time frame, S100A9 inhibited the M-CSF-mediated induction of RANK, which we found to be associated with changes in various histone marks at the epigenetic level. This S100A9-induced reduction in RANK could be partially reversed by blocking TNFα using etanercept, but not by blocking interleukin-1 (IL-1) with the IL-1 receptor antagonist. Conclusion Whereas S100A8/A9 was previously shown to stimulate the resorptive capacity of mature osteoclasts, we here show that early S100A9 stimulation impedes monocyte-to-osteoclast differentiation via reduction of RANK expression that is partially TNFα-mediated. This suggests that the timing of exposure to S100A8/A9 is an important determinant for monocyte-to-osteoclast differentiation. Disclosure of Interests Martijn van den Bosch: None declared, Irene Di Ceglie: None declared, Arjen Blom: None declared, Robab Davar: None declared, Colin Logie: None declared, Ehsan Habibi: None declared, Johannes Roth: None declared, Thomas Vogl: None declared, Carl Goodyear Grant/research support from: AstraZeneca, BMS, Celgene, Janssen, MedAnnex, Pfizer and UCB, Speakers bureau: Abbvie, Peter van der Kraan: None declared, Peter van Lent: None declared