Background: Systemic Sclerosis (dcSSc) is an autoimmune disorder that affects the connective tissue. It is characterized by the thickening of the skin, which can lead to the fibrosis of internal organs. Although the overactivation of the immune system and increased proinflammatory cytokine production can explain the pathophysiology of this disease to a certain extent, the mechanisms leading to this are not well understood. Checkpoint inhibitory molecules function as physiological brakes in the immune system and help to maintain homeostasis. Aberrant checkpoint inhibitory pathways are often observed in patients with dcSSc. Lymphocyte activation gene-3 (LAG-3) is a late checkpoint inhibitory receptor that is mainly expressed by T cells and is involved in T cell inhibition. In this study, we focused on the role of LAG-3 in dcSSc and the effect of a LAG-3 agonist on immune cells from these patients. Objectives: This study aims to analyse the role of LAG-3 dcSSc and the influence of a LAG-3 Agonistic antibody in influencing inflammation and fibrosis. Methods: Plasma LAG-3 levels of 36 patients with dcSSc were compared to 20 healthy controls. PBMC’s from eight dcSSc patients and four Healthy Controls were analysed by flow cytometry for the surface expression of LAG-3 on CD4+ and CD8+T cells after stimulation with CD3/CD28 for 24 hours. Eight dcSSc PBMC’s were stimulated for 48 h with a LAG-3 Agonistic antibody and an isotype control. The harvested supernatants were analysed using a multiplex proinflammatory cytokine ELISA. Results: Patients with dcSSc had decreased soluble levels of LAG-3 compared with healthy controls (p <0.0001). Increased surface expression of LAG-3 was observed in CD4+ T cells(p=<0.05) and CD8+ T cells(p=<0.005) in patients with dcSSc compared to healthy control PBMCs. When treated with a LAG-3 agonistic antibody (LAG-3 Ag), PBMC monocultures from dcSSc patients significantly downregulated the production of IFN gamma, IL-2, IL-4 and TNF alpha and upregulated the production of IL-10 as compared to non-treated controls(NT) (*=p<0.05,**=p<0.005). The Isotype control did not show a similar effect. Conclusion: LAG-3 is immune-protective in Systemic Sclerosis and its agonistic antibody could be used as a potential treatment modality. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.Figure 1

Background: Systemic sclerosis (SSc) is an autoimmune disease characterised by the production of autoantibodies, which leads to fibrosis and finally damage to various tissues and organs. Recent studies have demonstrated activation and aberrant persistence of M2-like macrophages in the disease tissues, implicating these cells as a source of pro-fibrotic and inflammatory mediators. However, the interplay between macrophages and autoantibodies remains largely unexplored. In this study, we aimed to investigate the role of disease-specific immunoglobulin G (IgG) autoantibodies, immune complexes, and plasma, on macrophage polarisation. We focused on the anti-topoisomerase (ATA) positive disease as a severe subgroup with strong M2 macrophage signature. Objectives: We aimed to test whether IgG purified from ATA positive SSc plasma, ATA:topoisomerase immune complexes or else unfractionated ATA SSc plasma affected the polarization state of healthy control or SSc disease macrophages. Methods: SSc and healthy control macrophages (both n=4) were obtained from blood monocytes following negative selection with RosetteSep and culture in X-vivo10 serum free media with the addition of M-CSF 4ng/ml for 7 days. IgG were isolated from ATA positive SSc plasma samples using Protein A IgG Purification Columns (Thermo Scientific) and added at 0.1, 10 and 100μg/ml to cultured macrophages. In other experiments the effects of ATA were compared with co-addition of human recombinant Topoisomerse I (Topo-I)(abcam, purity > 95%)in 4 conditions, media only, ATA only, ATA+Topo-I, and Topo-I only. Final concentrations in each well for ATA and Topo-I were 100μg/ml and 20ng/ml, respectively. Recombinant Topo-I was mixed with ATA or media solution before the treatment and incubated at 37°C and 5% CO2 for 2hrs to allow complex formation. qPCR assays for CD206 (mannose receptor), CD80 (co-stimulatory) and MerTK (regulatory/efferocytosis) were used to evaluate the polarization state and TBP used as the reference gene. In other experiments unfractionated ATA positive SSc plasma was added to media at 5% to stimulate the cells. Results: In healthy control macrophages ATA addition was found to decrease CD206 and CD86 but to enhance IL-10 and MerTK expression levels. Similar trends were seen in SSc macrophage lines, consistent with some reduction in M2a properties and enhancement of M2c by the effect of ATA. Different effects were seen when cells were stimulated by ATA:Topo-I immune complexes, leading to no change in CD206, but marked enhancement of CD86 (p<0.031) and reduction of IL-10 (p<0.019) and less MerTK (p<0.067), consistent with M2b polarization. Finally, addition of 5% SSc plasma led to complex activation of healthy control macrophages as indicated by enhanced CD206 (p<0.05), enhanced CD86 (p<0.051) and greatly enhanced IL-10 (p<0.016) and MerTK (p<0.0049), with similar trends in SSc macrophages excepting that IL-10 was not induced. (SSc macrophage data Figure 1A). Conclusion: Taken together these results indicate that SSc ATA IgG autoantibodies are capable of inducing changes in macrophage activation state and exert differential effects when added in isolation or as immune complexes. ATA in isolation appeared to induce an M2c like phenotype normally associated with phagocytosis and resolution. When added as immune complexes the ATA:Topo-I induced CD86 consistent with an effector/inflammatory M2b phenotype. By contrast, adding unfractionated ATA positive SSc plasma, a known source of disease-related cytokines and growth factors, induced more complex activated M2-like changes. Failure to induce IL-10 was a major differences in SSc macrophages, as well as higher basal CD206. Taken together the results indicate a major influence of ATA on macrophage activation states, which are differentially induced dependent on presence of the Topoisomerase antigen. The formation of immune complexes with Topoisomerase released by damaged and apoptotic cells might have an important modulating effect on the induction of macrophages by ATA. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Sandra Lopez Garces: None declared, Siyu Zhang: None declared, Henry Lopez CEO of Riptide Bioscience Inc, David Abraham Research grants received from UCB, Christopher P Denton: None declared, Teresa Collins: None declared, Jennifer Cross: None declared, Bahja Ahmed Abdi: None declared, Richard Stratton: None declared.Figure 1

Interstitial lung disease (ILD) is the most common cause of death in patients with Systemic sclerosis (SSc). Nearly all SSc-ILD patients test positive for antinuclear antibodies, but their autoantigen specificity is known only in a proportion. A large subset of patients with autoantibodies of unknown specificity, have the second highest rate of ILD progression after the Scl70+ group (Nihtyanova et al. doi: 10.1002/art.41153). Identification of patients at higher risk of ILD progression is key to optimal treatment and monitoring. A panel of 64 auto-antibodies was measured in the serum of 288 UK-based SSc patients (243 with ILD), and 50 controls, by Luminex immunoassay. Presence of each auto-antibody was defined as a mean fluorescence intensity >98% of the controls. Associations with the presence of ILD were assessed by Fisher’s exact test, and survival by Cox regression. Novel auto-antibodies to CBLC (6.7% in SSc-no ILD vs 0.8% in SSc-ILD, p=0.03), CD47 (15.6% vs 4.1%, p=0.008), HNRNPA1 (11.1% vs 2.9%, p=0.03), and KDM6B (24.4% vs 4.4%, p=0.002) were associated with absence of ILD. Presence of auto-antibodies to TRIM21 (Ro52), observed in 113 (39.2%) of our SSc cohort, was associated with increased mortality (median survival 12.0 vs 16.3 years), even after multivariable analysis, adjusting for age, gender, smoking history, and baseline lung disease severity (composite physiological index) in all patients (HR:1.48, 95%CI: 1.04-2.10, p=0.029), and in those with ILD (HR:1.56, 95%CI:1.07-2.28, p=0.02). In SSc patients, auto-antibodies, including of novel autoantigen specificity included in this study, can improve disease subgrouping and highlight pathogenetic pathways linked to more progressive ILD.

Background Pulmonary fibrosis is one of the major manifestations in Systemic Sclerosis (SSc) associated with high mortality. Mesenchymal transformation of the airway epithelial cells has been implicated as one of the causes for developing pulmonary fibrosis. Though several animal models shed light towards some of these aspects, an in vitro airway epithelial model would provide a novel experimental platform for the understanding and molecular and genetic changes that occur in SSc associated pulmonary fibrosis. Objectives To establish a functional model for airway epithelium from patient with diffuse cutaneous SSc (dSSc)and healthy volunteers derived nasal stem cells. Subsequently to induce Epithelial Mesenchymal transformation (EMT). Methods Nasal stem cells harvested from healthy volunteers(HV) and dSSc patients were differentiated into ciliated airway epithelium in an Air -Liquid Interface (ALI) using a transwell system. 4 HV cultures were then stimulated with TGF beta (5ug/ml) for 10 days at a basal stage and when differentiated. Markers of mesenchymal transformation including loss of E cadherin, and gain of N cadherin, fibronectin and vimentin were analysed by flow cytometry and image stream, and mean expression intensities given as (MFI). Secreted Type 1 collagen and fibronectin were measured by ELISA. Results Ciliated epithelial cultures could successfully be established from nasal stem cells (Figure 1). TGF beta induced a phenotypic change in the epithelial cells towards a mesenchymal one in HV cultures. This was observed by significantly increased expression of fibronectin and vimentin and loss of expression of E cadherin on the ciliated cells with 7 days of stimulation with TGF beta at a basal stage (Figure 1b). When cells, stimulated with TGF beta for 7 days, were analysed at Day 35 a similar trend was seen in their Delta MFI (Figure 1c). Stimulating the ALI cultures with TGF beta for 20 days completely repressed epithelial cell growth and disrupted their microstructure. Figure 1. Conclusion This novel ALI differentiated Airway epithelial model serves as a functional organoid to test various pulmonary manifestations of Systemic Sclerosis. The ability to induce Epithelial Mesenchymal Transformation of these cultures provides a proof of concept for TGF beta mediated fibrosis in dSSc. Moreover, this model can be utilized to explore, at the cell and molecular level, the impact of various autoantibodies and therapeutics on epithelial cells. References [1]Mehmet Kesimer,1 Sara Kirkham,2 Raymond J. Pickles,3 Ashley G. Henderson,4 Neil E. Alexis,5 Genevieve DeMaria,1 David Knight,2 David J. Thornton,2 and John K. Sheehan1 Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?; Am J Physiol Lung Cell Mol Physiol 296: L92–L100, 2009 Disclosure of Interests None declared.

Background: Systemic sclerosis (scleroderma, SSc) is a heterogeneous disease in which clinical outcomes vary widely. Predicting outcomes on an individual basis remains challenging despite progress made through autoantibody analysis and gene expression profiling. Effective targeted therapies are evolving and accurately predicting outcomes is important to enable patient stratification for therapy. Chromatin Conformation Signature (CCS) profiling of peripheral blood for systemic epigenetic deregulations could be used for such a purpose. The EpiSwitch platform offering high throughput and resolution chromosome conformation (3C) capture detects significant regulatory changes in 3D genome architecture and maps long range interaction between distant genomic locations. This then reveals the spatial disposition and physical properties of the chromosome, such as chromatin loops and inter-chromosomal connections, which have a role in network organization and genetic epistasis controlling gene expression. EpiSwitch automated platform has been successfully utilised in patient stratification in RA, MS and other indications. This methodology could be applied to patients with SSc to identify CCS associated with different phenotypes and may ultimately be used to stratify and identify patients into pathogenic subtypes. Objectives: We aimed to determine significant CCSs associated with early and late phenotypes of SSc. Methods: The EpiSwitch-based chromosome conformation capture (3C) method was applied to blood samples from early phenotype, and late phenotype SSc patients. Intact nuclei were isolated from peripheral blood mononuclear cells and subjected to formaldehyde fixation resulting in crosslinking between physically touching segments of the genome via contacts between their DNA bound proteins. For quantification of cross-linking frequencies, the cross linked DNA was digested and then subjected to ligation. Cross-linking was then reversed and individual ligation products detected and quantified by EpiSwitch custom oligo array annotated across the whole genome to the anchoring sites of 3D genome architecture. Results: 7 significant CCSs were found over the HLA-C, HLA-B and TNF regions on Chromosome 6 in the early phenotype. The top 8 pathways for genetic locations associated to the CCSs are shown in Table 1. Table 1. Top 8 pathways for genetic locations associated to significant CCS for the early phenotype. GeneSet 1 Natural Killer cell mediated cytotoxicity 2 Immunoregulatory interations between a lymphoid cell and a non-lymphoid cell 3 Antigen Processing & presentation 4 Phagosome 5 Graft versus host disease 6 Type 1 diabetes mellitus 7 Osteoclast differentiation 8 Class 1 MHC mediated antigen processing & presentation 2 significant CCSs were found centred around the IFNG region of chromosome 12 in the late phenotype. The top 8 pathways for genetic locations associated to significant CCSs are shown in Table 2. Table 2. Top 8 pathways for genetic locations associated to significant CCS for the late phenotype. GeneSet 1 Surfactant metabolism 2 IL12 signalling mediated by STAT4 3 Protein digestion & absorption 4 Calcineruin regulated NFAT dependent transcription in lymphocytes 5 Transcriptional misregulation in cancer 6 Kaposi’s sarcoma associated herpes virus infection 7 IL2 mediated signalling events 8 Inflammatory bowel disease Conclusion: Significant CCSs, as part of 3D genomic regulatory control, and their associated pathways for the genetic locations, were identified in both late and early phenotypes. There were distinct CCSs in the early phenotype compared to the late suggesting the CCSs change as the disease progresses and varies between phenotypes. If CCSs could be linked to each clinically defined subgroup across a SSc cohort they could be used as a biomarker tool to predict outcome and progression in patients. Disclosure of Interests: Megan Galloway: None declared, Ewan Hunter: None declared, Alexandre Akoulitchev: None declared, Shivanee Vigneswaran: None declared, Bahja Abdi: None declared, Christopher Denton Grant/research support from: GlaxoSmithKline, Inventiva, CSF Behring, Consultant of: Roche-Genentech, Actelion, GlaxoSmithKline, Sanofi Aventis, Inventiva, CSL Behring, Boehringer Ingelheim, Bayer, David Abraham: None declared, Richard Stratton: None declared

Background: Recent studies suggest dysregulation in T cell activation in systemic sclerosis (SSc). Co-inhibitory-receptors (Co-IRs) such as TIM-3, PD-1 and LAG-3 play a crucial role in controlling excessive T cell activation and in maintaining immune homeostasis. Engagement of these receptors by their ligand’s limits cytokine production in response to TCR or activating NK receptor stimulation and hence limit tissue damage from excessive immune activation. However, chronically increased expression of multiple Co-IRs is a hallmark of immune exhaustion. We evaluate the role of these soluble Co-IRs in diffuse SSc (dcSSc). Objectives: Establish the role of CiR and their ligands in diffuse systemic sclerosis. Understand how immune regulatory mechanisms influence the development of fibrosis. Provide a better understanding of the disease and fibrosis in general. Methods: PBMC’s(Peripheral blood mononuclear cells) and dermal fibroblasts from SSc patients were isolated and investigated for markers of T cell inhibition. These cells were analysed using flow cytometry in a 10 colour panel. Cells were stained for PD1, TIM3, TIGIT, LAG3, CD3, CD8, CD4 and CD19 along with a Live/dead marker. Co-cultures of fibroblasts and PBMCs will be setup, and treated with various drugs that act on the Co-IRs. Results: The proportion of CD4+ T cells expressing PD1 were markedly increased in SSc patients compared to healthy volunteers and Rheumatoid Arthritis patients. There was increased expression of both TIGIT and TIM3 in the CD4+ T cells. (Figure 1) Similarly, the co-expression of these receptors on the CD4+ T cell population was elevated compared to healthy volunteers. (figure 2) Conclusion: Soluble co-inhibitors are differentially expressed in early dcSSc compared to healthy volunteers and other autoimmune diseases. Our preliminary data indicates that these co inhibitors could play an important role in unravelling the pathogenesis of systemic sclerosis. Inhibition or activation of these receptors through different treatment modalities can be utilized as a novel patient centric treatment strategy. References: [1]Fukasawa, T., Yoshizaki, A., Ebata, S., Nakamura, K., Saigusa, R., Miura, S., … Sato, S. (2017). Contribution of Soluble Forms of Programmed Death 1 and Programmed Death Ligand 2 to Disease Severity and Progression in Systemic Sclerosis. Arthritis & Rheumatology , 69 (9), 1879–1890. [2]Greisen S, Rasmussen T, Stengaard-Pedersen K, Hetland M, Hørslev-Petersen K, Hvid M, et al. Increased soluble programmed death-1 (sPD-1) is associated with disease activity and radiographic progression in early rheumatoid arthritis. Scand J Rheumatol 2014; 43:101-8. [3]de Paoli, F., Nielsen, B., Rasmussen, F., Deleuran, B., & Søndergaard, K. (2014). Abatacept induces clinical improvement in patients with severe systemic sclerosis. Scandinavian Journal of Rheumatology , 43 (4), 342–345. [4]Kwon, B. (2010). Intervention with costimulatory pathways as a therapeutic approach for graft-versus-host disease. Experimental and Molecular Medicine . Nature Publishing Group. Acknowledgments: FOREUM: Foundation of Research in Rheumatology Disclosure of Interests: None declared

Background: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by microangiopathy and fibrosis. In physiological wound healing, fibroblasts are transiently activated for tissue repair. In contrast, fibroblasts are persistently activated during fibrosis and thus resulted in progressive matrix deposition and tissue remodeling. However, the pathogenesis of the fibrotic process is not fully understood. Objectives: We aimed to identify molecules that play a role in chronically activated fibroblasts. Methods: To identify molecules specifically upregulated in human fibrotic fibroblasts, RNA-sequencing was performed. Identified adaptor proteins were further validated in skin biopsy samples of patients with limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc), and evaluated correlation between expression levels and clinical parameters. Respective overexpression and siRNA knockdown were further addressed in vitro . Functional effects were assessed by qPCR, hydroxyproline, proliferation and migration assays. Mouse models of systemic sclerosis were used to functionally validate adaptor proteins in vivo . Results: We identified adaptor proteins as significantly upregulated molecules in chronically active fibroblasts of skin biopsy samples from SSc patients compared to fibroblasts from healthy controls. Expression levels were correlated with the modified Rodnan skin score in the skin of SSc patients. We observed higher expression levels also in the mouse model of topoisomerase I induced skin fibrosis. This result was also observed in bleomycin induced lung fibrosis model suggesting important functions of adaptor proteins during fibrotic tissue remodeling across different organs. Fibroblast-specific knockout resulted into significantly attenuated bleomycin-induced fibrosis. Upon bleomycin challenge, hydroxyproline content was diminished in mice with genetic deficiency of adaptor proteins. In addition, COL1A1, COL1A2 and Lum mRNAs and also the number of myofibroblasts were significantly lower in knockout mice compared to wild type mice. In vitro , knockdown of adaptor proteins resulted into a significant alteration of the migratory potential of fibroblasts. Conclusion: Our results demonstrate that adaptor proteins play an essential role in the pathogenesis of systemic sclerosis. Understanding the molecular mechanism of adaptor proteins may lead to a novel therapeutic intervention in human SSc and related disorders.

Journal of the European Academy of Dermatology and VenereologyVolume 34, Issue 5 p. e236-e238 Letter to the Editor Whole blood gene expression profiling distinguishes systemic sclerosis-overlap syndromes from other subsets P. Moinzadeh, Corresponding Author P. Moinzadeh pia.moinzadeh@uk-koeln.de orcid.org/0000-0002-8784-8615 Department of Dermatology and Venerology, University of Cologne, Cologne, GermanyCorrespondence: P. Moinzadeh. E-mail: pia.moinzadeh@uk-koeln.deSearch for more papers by this authorP. Frommolt, P. Frommolt CECAD University of Cologne, Cologne, GermanySearch for more papers by this authorM. Franitza, M. Franitza Cologne Center for Genomics, University of Cologne, Cologne, GermanySearch for more papers by this authorM. R. Toliat, M. R. Toliat Cologne Center for Genomics, University of Cologne, Cologne, GermanySearch for more papers by this authorK. Becker, K. Becker Cologne Center for Genomics, University of Cologne, Cologne, GermanySearch for more papers by this authorP. Nürnberg, P. Nürnberg CECAD University of Cologne, Cologne, Germany Cologne Center for Genomics, University of Cologne, Cologne, GermanySearch for more papers by this authorS. I. Nihtyanova, S. I. Nihtyanova Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Hospital, London, UKSearch for more papers by this authorM. Ahrazoglu, M. Ahrazoglu Department of Dermatology and Venerology, University of Cologne, Cologne, GermanySearch for more papers by this authorD. Belz, D. Belz Department of Dermatology and Venerology, University of Cologne, Cologne, GermanySearch for more papers by this authorN. Hunzelmann, N. Hunzelmann Department of Dermatology and Venerology, University of Cologne, Cologne, GermanySearch for more papers by this authorD. Abraham, D. Abraham Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Hospital, London, UKSearch for more papers by this authorV. H. Ong, V. H. Ong Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Hospital, London, UKSearch for more papers by this authorL. Mouthon, L. Mouthon National Reference Centre for Scleroderma and Systemic Vasculitis (LM, LG), Université Paris Descartes, Cochin Hospital, Paris, FranceSearch for more papers by this authorR. Hesselstrand, R. Hesselstrand Department of Rheumatology, Lund University, Lund, SwedenSearch for more papers by this authorC. P. Denton, C. P. Denton Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Hospital, London, UKSearch for more papers by this authorT. Krieg, T. Krieg Department of Dermatology and Venerology, University of Cologne, Cologne, GermanySearch for more papers by this author P. Moinzadeh, Corresponding Author P. Moinzadeh pia.moinzadeh@uk-koeln.de orcid.org/0000-0002-8784-8615 Department of Dermatology and Venerology, University of Cologne, Cologne, GermanyCorrespondence: P. Moinzadeh. E-mail: pia.moinzadeh@uk-koeln.deSearch for more papers by this authorP. Frommolt, P. Frommolt CECAD University of Cologne, Cologne, GermanySearch for more papers by this authorM. Franitza, M. Franitza Cologne Center for Genomics, University of Cologne, Cologne, GermanySearch for more papers by this authorM. R. Toliat, M. R. Toliat Cologne Center for Genomics, University of Cologne, Cologne, GermanySearch for more papers by this authorK. Becker, K. Becker Cologne Center for Genomics, University of Cologne, Cologne, GermanySearch for more papers by this authorP. Nürnberg, P. Nürnberg CECAD University of Cologne, Cologne, Germany Cologne Center for Genomics, University of Cologne, Cologne, GermanySearch for more papers by this authorS. I. Nihtyanova, S. I. Nihtyanova Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Hospital, London, UKSearch for more papers by this authorM. Ahrazoglu, M. Ahrazoglu Department of Dermatology and Venerology, University of Cologne, Cologne, GermanySearch for more papers by this authorD. Belz, D. Belz Department of Dermatology and Venerology, University of Cologne, Cologne, GermanySearch for more papers by this authorN. Hunzelmann, N. Hunzelmann Department of Dermatology and Venerology, University of Cologne, Cologne, GermanySearch for more papers by this authorD. Abraham, D. Abraham Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Hospital, London, UKSearch for more papers by this authorV. H. Ong, V. H. Ong Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Hospital, London, UKSearch for more papers by this authorL. Mouthon, L. Mouthon National Reference Centre for Scleroderma and Systemic Vasculitis (LM, LG), Université Paris Descartes, Cochin Hospital, Paris, FranceSearch for more papers by this authorR. Hesselstrand, R. Hesselstrand Department of Rheumatology, Lund University, Lund, SwedenSearch for more papers by this authorC. P. Denton, C. P. Denton Centre for Rheumatology and Connective Tissue Diseases, UCL Medical School, Royal Free Hospital, London, UKSearch for more papers by this authorT. Krieg, T. Krieg Department of Dermatology and Venerology, University of Cologne, Cologne, GermanySearch for more papers by this author First published: 15 January 2020 https://doi.org/10.1111/jdv.16198Citations: 3Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume34, Issue5May 2020Pages e236-e238 RelatedInformation

The onset of inflammation, hypoxia or shear stress within blood vessels can result in endothelial-to-mesenchymal transition (EndoMT), a disease-associated process where endothelial cells (ECs) downregulate endothelial markers and acquire mesenchymal features. EndoMT is observed in patients with scleroderma-associated pulmonary hypertension (SSc-PAH), which have the highest mortality amongst all the scleroderma patient subgroups. The homeobox transcriptional factor NKX2-5 is fundamental for cardiovascular development. However, NKX2-5 expression has not been reported yet in ECs of adult pulmonary blood vessels. To investigate the role of NKX2-5 in the pulmonary endothelium of SSc-PAH. Human pulmonary artery endothelial cells (HPAECs) were treated with a cocktail of TGF-β (5 ng/mL), TNF-α (5 ng/mL), and IL-1β (0.1 ng/mL) for 5 days. Immunofluorescence was used to detect NKX2-5 and other markers in ECs. Western blotting and qPCR evaluated, respectively, protein and gene expression. Lentiviral transduction forced NKX2-5 expression in the cells. Transendothelial electrical resistance (TEER) measurements evaluated endothelial barrier function. Pharmacological inhibition was performed to determine the pathways that lead to NKX2-5 activation. Casein kinase 2 (CK2)-inhibition (CX4945) of a chronic hypoxia mouse model of PAH was used to assess right ventricular systolic pressure (RVSP). Immunofluorescence showed a strong expression of NKX2-5 in the endothelium of SSc-PAH human lungs (p<0.0001). Western blot analysis demonstrated a 5.3-fold downregulation of CD31 (p<0.001), and an increased production of NKX2-5 (5.6-fold, p<0.0001) and of Procollagen I (12-fold, p=0.0009) after 5 days of cytokine stimulation on HPAECs. Relative mRNA expression has shown a 3-fold gene downregulation of CD31 (p=0.0002) and a reduction of VE-Cadherin (2.3-fold, p=0.0008) and of vWF (10.4-fold, p=0.003) in EndoMT, whereas gene expression of COL1α2 (8.5-fold, p<0.0001) and of NKX2-5 (1.5-fold, p=0.003) were upregulated. Immunofluorescence of cells has revealed a decreased VE-Cadherin expression concomitant with upregulation of NKX2-5 in EndoMT cells. Forced expression of NKX2-5 downregulated endothelial markers and endothelial barrier function was impaired whereas proliferation rate of cells was increased. Inhibition of PI3K, ERK5, ALK5 and CK2 reduced NKX2-5 protein expression within cells. CK2-inhibited mice under hypoxia conditions resembled the normoxia mice group by normalising RVSP. HPAECs undergoing EndoMT express NKX2-5 in vitro and in vivo, via mediation of CK2, TGF-β, ERK5 and PI3K signalling. NKX2-5 downregulates key adherence junctional proteins, disrupting endothelial barrier function. This study highlights the involvement of NKX2-5 in EndoMT and in endothelial dysfunction, leading to vascular disease progression in SSc-PAH. British Heart Foundation, Arthritis Research UK, Scleroderma Research UK and Royal Free Hospital Charity

Purpose: CCN2 is a matricellular protein which has been shown to be heavily involved in several complex biological processes including angiogenesis, fibrosis, and chondrogenesis. CCN2 is known to be expressed in both healthy and osteoarthritic cartilage, and involved in the production of the major cartilage matrix proteins; aggrecan and collagen type II. CCN2 null mice have been reported to exhibit a range of skeletal dysmorphisms, highlighting the importance of CCN2 in regulating matrix formation and turnover. The aim of this study was to determine the function of CCN2 in chondrocytes postnatally, in models of trauma-induced osteoarthritis (OA). Methods: Three double transgenic mouse models were generated; two inducible CCN2 knockout (KO) lines and one inducible CCN2 overexpressing (O/E) line. Both CCN2 KO lines contained Aggrecan CreERT2 enhancers which enabled the deletion of CCN2 from different populations of chondrocytes, whilst the O/E line contained an UbcCCN2-IRES-LacZ transgene enabling CCN2 O/E in all chondrocytes. CCN2 deletion and overexpression was induced following tamoxifen treatment in males aged 8 weeks. OA was induced either through surgical injury (transection of the medial meniscus and medial menisco-tibial ligament), or non-invasively by applying a controlled loading regimen to the tibio-femoral joint at 10 weeks of age. Knee joints were harvested and fixed, scanned with μCT, and processed for histology. Sections were stained with Toluidine Blue and scored for articular cartilage degeneration using the OARSI grading system. Results: In the surgical model of OA, cartilage degeneration was most severe on the medial side of the joint, however no significant differences were observed between lesion severity in wildtype (WT) and KO at any of the time-points analysed (2, 4, and, 8 weeks post-surgery). In the non-invasive model of OA lesion severity was most severe on the lateral femur in both the KO and O/E lines. As with the surgical model, no differences in lesion severity were observed between WT / KO and WT / O/E when analysed 6 weeks post-loading. μCT analysis of trabecular bone BV/TV and joint space in both models showed no effect of CCN2 KO and O/E between WT and experimental animals. Conclusions: CCN2 did not appear to play a protective role in the development of OA irrespective of whether the damage was induced surgically or non-invasively. The progression and severity of OA in both models, at all time-points, suggests the expression of CCN2 specifically in chondrocytes is not enough to protect articular cartilage from the degeneration associated with trauma-induced OA.

Background Pulmonary fibrosis is a major cause of mortality in scleroderma (SSc) and Idiopathic Pulmonary Fibrosis (IPF). Fibrosis is driven by Inappropriate myofibroblast differentiation and persistence. Understanding this process, is vital for developing an effective treatment. Angiotensin II, is implicated in fibroblast activation in the heart and kidney, through interactions with growth factors (e.g. EGF and TGFβ). Objectives We examined the role of Angiotensin II in myofibroblast activation in the lung. Methods Lung fibroblasts were isolated from SSc, IPF, or control patient lungs (6 each). Fibroblasts were also cultured from PTEN null and wild-type mice. Protein expression after angiotensin II treatment (AngII) was investigated by western blotting. Myofibroblast differentiation and function was assayed through the contraction of 3D collagen gels and scratch migration assays. The signalling pathways involved were dissected using specific inhibitors: PI3-kinase/AKT (wortmannin, LY294002), TGFβ (1d11 neutralising antibody, SB431542 ALK5 inhibitor) Ataxia-Telangiectasia Mutated (ATM – Ku55933), AngII (Losartan). Results SSc and IPF lung fibroblasts showed increased AKT phosphorylation and suppressed PTEN expression (p<0.05). Their phenotype was more myofibroblast-like, with higher αSMA expression (p<0.05), increased collagen gel contraction (control; 207±14 vs SSc; 93±15 vs IPF 91±21, p<0.05), and enhanced migratory capacity (p<0.05). PTEN-null fibroblasts showed a similar phenotype. AngII treatment activated AKT, suppressed PTEN and induced myofibroblast differentiation in normal lung fibroblasts. In both AngII-treated and PTEN lung fibroblasts AKT activation required the ATM kinase. Inhibition of AKT either with PI3K or ATM inhibitor abrogated these effects. The increased expression of Myofibroblast-related genes after AngII treatment, was also blocked by inhibition of TGFβ with a neutralising antibody or an ALK5 inhibitor. AKT phosphorylation on the other hand was only partially blocked was partially blocked by TGFβ inhibition. Conclusions Our data demonstrate for the first time that AngII signals via the ATM kinase, which together with PTEN suppression are essential for the activation of AKT by AngII. AngII promotes myofibroblast differentiation, by stimulating the fibroblast TGFβ autocrine loop through AKT. Our data shows that activation of AKT through ATM and PTEN, may serve as the molecular link between pulmonary hypertension and lung fibrosis in fibrotic diseases. Acknowledgements Arthritis Research UK, Royal Free Hospital Charity and Scleroderma Research UK. Disclosure of Interest None declared

Background Three prolyl 3-hydroxylase enzymes, LEPRE1, LEPREL1 and LEPREL2, are known to modify prolines in certain sequences in the C-terminal helical region of the polypeptide chains of procollagens converting them to 3-hydroxyproline residues. This modification appears to facilitate correct alignment of the chains in forming the triple helical domains of the procollagen molecules prior to secretion. Increased deposition of triple helical collagen and other extracellular matrix (ECM) proteins by activated fibroblasts underlies pathological fibrosis in systemic sclerosis (SSc), and may be dependent on prolyl 3-hydroxylase activity as a rate-limiting step. Objectives The objectives of this study were: 1) to screen candidate genes with large first introns containing regulatory elements for association with systemic sclerosis (SSc) through copy number variation (CNV), 2) to study the lead candidate gene LEPREL1 further, as a fibrosis-related factor in genetically modfied mice subject to bleomycin induced skin fibrosis, 3) to determine the levels of LEPREL1 protein in SSc and control fibroblasts and measure possible induction by profibrotic factors, and 4) measure the effect of LEPREL1 gene editing on triple helical collagen secretion by SSc disease fibroblasts. Methods DNA samples from SSc patients and controls were assayed by qPCR for CNVs within regulatory regions of 40 candidate genes. The lead candidate, LEPREL1 was studied further through tagging SNP analysis, of rs7612998, rs1447936, s1018343, s696065, in 564 SSc and 627 controls. In wild type (WT), GPVI -/-single knockout (SKO) or LEPREL1-/- GPVI -/-double knockout (DKO) mice skin fibrosis was initiated by daily subcutaneous bleomycin for 14 days (LEPREL1-/- embryonically lethal due to placental thrombosis). Skin fibrotic lesions sampled on day 21 were analysed by histology and picrosirius red stain. SSc and control skin fibroblasts grown from forearm skin biopsies, were cultured at passage 3–5, with or without the pro-fibrotic TGFbeta, or with or without estradiol in cells grown in oestrogen depleted conditons (no phenol red). LEPREL1 was assayed by Western blot. CRISPR/Cas9 was used to edit the LEPREL1 gene in SSc skin fibroblasts. Results Initial screening of candidate genes revealed a weak statistical association between a first intron CNV nsv514192 and SSc susceptibility in males (p<0.028). However SNP analysis demonstrated that a haplotype, identified as CTAA across the 4 SNPs, was associated with increased risk of SSc development (OR 3.45, p<0.0023). This allele opens a FOXA1 site in the first intron and was seen predominantly in females. In cultured dermal fibroblasts LEPREL1 protein levels were raised in SSc samples and induced to SSc levels in control fibroblasts by culture with TGFbeta. Estradiol also induced LEPREL1 and collagen I protein. In genetically modified LEPREL1 knockout mice, resistance to bleomycin skin fibrosis was seen (figure 1). Silencing of LEPREL1 in SSc fibroblasts by CRISPR/Cas9 preferentially reduced triple helical collagen I secretion. Conclusions The prolyl 3-hydroxylase LEPREL1 is confirmed as a fibrosis-related gene associated with SSc susceptibility. Small molecule inhibitors of this endoplasmic reticulum enzyme may limit the overproduction of collagen as a therapy for fibrosis. Disclosure of Interest None declared

BackgroundChronic and exaggerated fibroblast activation is a central hallmark of Systemic Sclerosis (SSc) fibrotic disease and results in a high morbidity and mortality. Epigenetic changes might play important roles in mediating chronic fibroblast activation. Trimethylation of H3 at lysine residue K27 (H3K27me3) is a repressive epigenetic mark that was recently identified as an important negative regulator of fibroblast activation [1]. Jumonji domain containing protein 3 (JMJD3) mediates H3K27me3–demethylation. JMJD3 inhibitors are being tested as therapeutic strategies in malignant diseases.ObjectivesThe aim of this study was to characterize the role of JMJD3 in fibrotic disease and to explore JMJD3 as a potential drug target in SSc.MethodsExpression analyses of JMJD3 were performed using qPCR, IF and Western blot. siRNA mediated knockdown and the pharmacologic H3K27me3-demethylase inhibitor GSKJ4 were used to target JMJD3. In vivo, we analyzed the effects of GSKJ4 in bleomycin-induced dermal fibrosis and in Topoisomerase-I-induced (TopoI) fibrosis. H3K27me3 levels at the Fra2 promotor were analyzed by CHIP.ResultsWe observed increased expression of JMJD3 in SSc skin compared to healthy controls. Fibroblast-specific overexpression of JMJD3 was also reflected in experimental fibrosis models. TGFβ upregulated JMJD3. Inhibition of JMJD3 increased H3K27me3 in vitro and in vivo. Inhibition of JMJD3 reverted the activated fibroblast phenotype in SSc fibroblasts and decreased the expression of contractile fibers and of α-smooth muscle actin. In addition, JMJD3 inhibition reduced the basal and TGFβ induced collagen secretion of SSc fibroblasts. JMJD3 regulated the TGFβ induced expression of Fra2. GSKJ4 reverted the TGFβ induced reduction of H3K27me3 at the Fra2 promotor. Moreover, the anti-fibrotic effects of JMJD3 inhibition were evened in Fra2 knockout fibroblasts. Overexpression of Fra2 in JMJD3-knockdown fibroblasts restored the profibrotic effect of JMJD3. In vivo, inhibition of JMJD3 ameliorated fibrosis in bleomycin- and TopoI- induced experimental fibrosis and reduced dermal thickening, hydroxyproline content and myofibroblast differentiation.ConclusionsWe present first evidence that JMJD3 contributes to the activated phenotype of SSc fibroblasts. TGFβ upregulated JMJD3. Inhibition of JMJD3 prevented the aberrant activation of fibroblasts in vitro and ameliorated dermal fibrosis in several mouse models in vivo. The profibrotic effects of JMJD3 might be mediated by reducing the H3K27me3 at the Fra2 promotor and consecutive overexpression of Fra2.ReferencesKramer, M., et al., Inhibition of H3K27 histone trimethylation activates fibroblasts and induces fibrosis. Ann Rheum Dis, 2013. 72(4): p. 614–20.Disclosure of InterestNone declared

Purpose: CCN2/CTGF (CCN family member 2/connective tissue growth factor, CCN2) is a matricellular protein that is expressed by chondrocytes in both healthy and osteoarthritic articular cartilage. It strongly promotes the production of cartilage matrix proteins, such as collagen type II and aggrecan, as well as stimulates chondrocyte proliferation, differentiation and maturation of growth-plate chondrocytes. The importance of CTGF on normal skeletal function is further demonstrated in CTGF null mice, which exhibit lethality at birth, possibly due to important skeletal defects. Indeed, these mice show severe chondrodysplasia caused by chondrogenesis defects, with decreased chondrocytes proliferation and aggrecan expression. In contrast, over-expression of CTGF lead to longer bones, increased accumulation of aggrecan and collagen type II in new born mice, as well as increased cell proliferation and enhanced chondrogenesis. Together, these studies confirm a crucial role of CTGF in regulating cartilage formation. The aim of this study is to determine the effect of CTGF deficiency in chondrocytes postnatally on osteoarthritis development. Methods: CTGF-Floxed mice were crossed with Tamoxifen-inducible Aggrecan-Cre-ER(T2) mouse line. CTGF gene deletion was induced specifically in chondrocytes following Tamoxifen treatment in male mice at 8 weeks of age. Destabilisation of the medial meniscus (DMM) model was used to promote posttraumatic OA at 10 weeks of age, and joints analysed at 4 and 8 weeks post surgery. Knee joints were dissected from skin and muscles, fixed in Formalin for 24h, and subsequently kept in 70% ethanol until required for analysis. Knees were scanned by micro-computed tomography (microCT, Skyscan 1176, Belgium; 5μm voxel size). Each dataset was analysed using 3D algorithms in CTAn (Skyscan, Belgium): subchondral and epiphyseal trabecular bone were manually separated and the regions of interests processed for measurement of subchondral bone thickness, trabecular Bone volume/Total volume and trabecular thickness. Joints were subsequently decalcified (EDTA) and processed for wax embedding. Serial coronal sections were stained with Safranin O at regular intervals across the joint, and scored for articular cartilage degradation severity, using the OARSI grading system (from grades 0–6). Results: Following DMM surgery, cartilage degeneration was more severe in the medial tibia of CTGF KO mice compared to WT at 4 weeks post surgery only, with maximum scores averaging 5.28 (±0.28 SEM) and 4 (±0; p=0.02), for KO and WT respectively. The lack of significant differences at 8weeks would be due to the high level of cartilage degradation in all groups. In contrast, bone analyses showed no significant differences in subchondral bone thickness and in trabecular bone BV/TV and trabecular thickness between WT and KO mice at either timepoints. Conclusions: Our preliminary analysis suggests that CTGF plays a role in limiting cartilage degeneration at 4 weeks, but did not modify bone changes in response to joint destabilisation. Further analysis of other joint tissues and pathways involved, and the addition of earlier timepoints may reveal further beneficial effects of CTGF of OA development and its mode of action.