A Psarras

Type I interferon–mediated autoimmune diseases: pathogenesis, diagnosis and targeted therapy

Type I interferons (IFN-Is) are a group of molecules with pleiotropic effects on the immune system forming a crucial link between innate and adaptive immune responses. Apart from their important role in antiviral immunity, IFN-Is are increasingly recognized as key players in autoimmune CTDs such as SLE. Novel therapies that target IFN-I appear effective in SLE in early trials, but effectiveness is related to the presence of IFN-I biomarkers. IFN-I biomarkers may also act as positive or negative predictors of response to other biologics. Despite the high failure rate of clinical trials in SLE, subgroups of patients often respond better. Fully optimizing the potential of these agents is therefore likely to require stratification of patients using IFN-I and other biomarkers. This suggests the unified concept of type I IFN-mediated autoimmune diseases as a grouping including patients with a variety of different traditional diagnoses.

A critical view on cardiovascular risk in systemic sclerosis

Systemic Sclerosis (SSc) is an autoimmune disorder characterized by microvascular injury and diffuse fibrosis of the skin and internal organs. While macrovascular disease and higher risk for cardiovascular events are well documented in other systemic rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus, the presence and extent of atherosclerosis among patients with SSc is yet to be established. Primary cardiac involvement, due to impairment of coronary microvascular circulation and myocardial fibrosis, considerably affects prognosis and life expectancy of individuals with SSc, representing one of the leading causes of death in this population. On the other hand the existence and prevalence of atherosclerotic coronary disease remains an issue of debate as studies comparing structural and morphological markers of atherosclerosis and cardiovascular events between SSc patients and the general population have yielded controversial results. The aim of this review is to summarize recent literature about the prevalence of cardiovascular disease in SSc, review the surrogate markers of CVD that have been evaluated and examine whether common pathogenic mechanisms exist between SSc and macrovascular disease.

A novel two-score system for interferon status segregates autoimmune diseases and correlates with clinical features

Measurement of type I interferon (IFN-I) has potential to diagnose and stratify autoimmune diseases, but existing results have been inconsistent. Interferon-stimulated-gene (ISG) based methods may be affected by the modularity of the ISG transcriptome, cell-specific expression, response to IFN-subtypes and bimodality of expression. We developed and clinically validated a 2-score system (IFN-Score-A and -B) using Factor Analysis of 31 ISGs measured by TaqMan selected from 3-IFN-annotated modules. We evaluated these scores using in-vitro IFN stimulation as well as in sorted cells then clinically validated in a cohort of 328 autoimmune disease patients and healthy controls. ISGs varied in response to IFN-subtypes and both scores varied between cell subsets. IFN-Score-A differentiated Systemic Lupus Erythematosus (SLE) from both Rheumatoid Arthritis (RA) and Healthy Controls (HC) (both p < 0.001), while IFN-Score-B differentiated SLE and RA from HC (both p < 0.001). In SLE, both scores were associated with cutaneous and hematological (all p < 0.05) but not musculoskeletal disease activity. Comparing with bimodal (IFN-high/low) classification, significant differences in IFN-scores were found between diagnostic groups within the IFN-high group. Our continuous 2-score system is more clinically relevant than a simple bimodal classification of IFN status. This system should allow improvement in diagnosis, stratification, and therapy in IFN-mediated autoimmunity.

Prediction of autoimmune connective tissue disease in an at-risk cohort: prognostic value of a novel two-score system for interferon status

Objective To evaluate clinical, interferon and imaging predictors of progression from ‘At Risk’ to autoimmune connective tissue diseases (AI-CTDs). Methods A prospective observational study was conducted in At-Risk of AI-CTD (defined as antinuclear antibody (ANA) positive; ≤1 clinical systemic lupus erythematosus (SLE) criterion; symptom duration <12 months and treatment-naïve). Bloods and skin biopsy (non-lesional) were analysed for two interferon-stimulated gene expression scores previously described (IFN-Score-A and IFN-Score-B). Forty-nine healthy controls (HCs) and 114 SLE were used as negative and positive controls. Musculoskeletal ultrasound was performed. Progression was defined by meeting classification criteria for AI-CTDs at 12 months. Results 118 individuals with 12-month follow-up were included. Of these, 19/118 (16%) progressed to AI-CTD (SLE=14, primary Sjogren’s=5). At baseline, both IFN scores differed among At-Risk, HCs and SLE groups (p<0.001) and both were elevated in At-Risk who progressed to AI-CTD at 12 months versus non-progressors, to a greater extent for IFN-Score-B (fold difference (95% CI) 3.22 (1.74 to 5.95), p<0.001) than IFN-Score-A (2.94 (1.14 to 7.54); p=0.018). Progressors did not have significantly greater baseline clinical characteristics or ultrasound findings. Fold difference between At-Risk and HCs for IFN-Score-A was markedly greater in skin than blood. In multivariable logistic regression, only family history of autoimmune rheumatic disease, OR 8.2 (95% CI 1.58 to 42.53) and IFN-Score-B, 3.79 (1.50–9.58) increased the odds of progression. Conclusion A two-factor interferon score and family history predict progression from ANA positivity to AI-CTD. These interferon scores may allow stratification of individuals At-Risk of AI-CTD permitting early intervention for disease prevention and avoid irreversible organ damage.

A9.06 Analysis of cell-specific interferon response in systemic lupus erythematosus using a novel flow cytometric assay

Background and objectives Type I interferons (IFN-I) have diverse effects on immune cell populations in SLE, Measuring IFN-I using whole blood interferon-stimulated gene (ISG) expression does not completely explain clinical features of SLE. Objective: develop a cell-specific assay using a surface protein encoded by an ISG (BST2). This would allow convenient analysis of IFN response in individual populations to improve immunophenotyping of SLE patients. Materials and methods PBMCs from 133 SLE patientsand 19 healthy controls (HC) were analysed by flow cytometry for cell surface BST2 protein on each immune cell subset. Cells were FACS-sorted into naïve and memory B-cells, plasmablasts, CD3+ T-cells, NK-cells and monocytes in 12 SLE patients and 16 healthy controls. Expression of BST2, as well as 32 other ISGs, were measured using qPCR. Results Analysis of sorted cells confirmed that surface BST2 is a valid cell-specific IFN assay. BST2 expression correlated with BST2 surface protein within each immune subset: naïve B-cells (r = 0.63, p = 0.009); memory B-cells (r = 0.78, p < 0.001); plasmablasts (r = 0.58, p = 0.018);NK cells (0.63, p = 0.008);T-cells (r = 0.61, p = 0.012); monocytes (r = 0.47, p = 0.064). We next used surface BST2 to compare IFN activity of each subset with clinical features in 133 patients. A significant correlation between the PBMC 33-gene IFN score and surface BST2 for each cell subset (all p < 0.001) confirmed validity of BST2 biomarker in this larger population as a measure of overall IFN status. BST2 was significantly higher in SLE than HC on naïve and memory B-cells (p = 0.004, p = 0.003), plasmablasts (p = 0.047), T cells (p = 0.043), but not different on monocytes (p = 0.406). Association of disease activity (total BILAG) with BST2 on naïve and memory B-cells (Tau-a = 0.23 and 0.22 respectively) was substantive and approximately twice as strong as monocytes and T-cells (Tau-a = 0.12 and 0.14). A similar pattern was seen for anti-dsDNA titre, with no association with monocyte BST-2 (Tau-a = 0.07) but a substantive association for memory B cell BST-2 (Tau-a = 0.18). Conclusion IFN-I response differs in cell subsets. This can be measured in a fast, cost-effective, convenient assay using flow cytometric analysis of surface BST2. Our results show that IFN activity measured on B cells is more clinically relevant than on other cell populations.

Publisher Correction: A novel two-score system for interferon status segregates autoimmune diseases and correlates with clinical features

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OP0180 Type i interferon is produced by non-haematopoietic tissue resident cells but not pdcs in pre-clinical autoimmunity and sle

Background Systemic Lupus Erythematosus (SLE) is characterised by persistently high type I interferon (IFN) activity. Plasmacytoid dendritic cells (pDCs) produce large amounts of IFNs in viral infection, although these immunogenic properties are usually strictly regulated. In vitro, pDCs are responsive to nucleic acids and they have therefore been postulated to be the main source of type I IFNs in SLE. However, their function is not fully established in human SLE. Objectives To investigate the dysregulated IFN axis in patients with pre-clinical autoimmunity and SLE. Methods Patients with SLE who met 2012 ACR/SLICC criteria were recruited. We also recruited healthy controls (HC) and therapy-naïve individuals presenting with ANA and 1–2 clinical symptoms, but not meeting ACR/SLICC criteria, of whom 17% progressed to SLE (At-Risk). IFN activity was evaluated by measuring a score of IFN-responsive genes in the PBMCs using TaqMan. pDCs were immunophenotyped as well as studied in vitro for production of proinflammatory cytokines and induction of T cell responses using flow cytometry. pDCs were sorted and sequenced using high-sensitive RNA sequencing. IFN expression was visualised in skin biopsies using in situ hybridisation. Keratinocytes were isolated from fresh skin biopsies and cultured in vitro; IFN production was measured by qPCR and ELISA. Results Most of the SLE and At-Risk patients had increased IFN activity, which correlated with disease activity and clinical features. In contrast, circulating pDCs were decreased in both SLE and At-Risk patients and their numbers did not correlate with any clinical features or IFN status. In vitro stimulation revealed that pDCs from SLE and At-Risk patients could not produce IFN-α and TNF-α upon stimulation with TLR9 or TLR7 agonists. In addition, they induced significantly less T cell activation and proliferation compared to HC pDCs. RNA-seq data analysis showed an upregulation of IFN-responsive genes in most of the SLE and At-Risk pDCs but not transcripts of any IFN subtypes. Other upregulated pathways were related to immune regulation and senescence. Phenotypically, SLE pDCs were characterised by upregulation of regulatory receptors and increased telomeric erosion. In situ hybridization revealed high IFN expression in the epidermis but not in lymphocyte-infiltrating areas of lesional biopsies from SLE patients. High expression of IFN was also observed in epidermis of At-Risk individuals without any signs of cutaneous inflammation. In vitro stimulation of freshly isolated keratinocytes also showed a notable increase in IFN production. Conclusions In SLE, non-haematopoietic tissue resident cells are a dominant source of IFN and this is present prior to clinically overt disease. Meanwhile, the professional IFN-producing pDCs have lost their immunogenic properties. These findings suggest an important role for tissue resident cells in autoimmunity and may facilitate novel therapeutic interventions. Disclosure of Interest None declared

SAT0019 Tnf-Α regulates plasmacytoid dendritic cells by suppressing ifn-Α production and enhancing th1 and th17 cell differentiation

Background Human plasmacytoid dendritic cells (pDCs) play a vital role in modulating immune responses. pDCs can produce massive amounts of type I IFNs in response to nucleic acids via toll-like receptors (TLRs) and they are known to possess weak antigen-presenting properties inducing CD4+ T cell activation. Previous data showed a cross-regulation between TNF-α and IFN-α but the effect of TNF-α on pDCs remains unclear. Objectives To investigate how TNF-α regulates the immune function of human pDCs. Methods Fresh PBMCs were treated with TNF-α, TLR7 and TLR9 synthetic agonists. pDCs were immunophenotyped using flow cytometry. RNA from sorted pDCs was extracted and sequenced using Smart-seq2 for sensitive full-length transcriptome profiling. For pDC/T cell co-culture, fresh or TNF-α-treated pDCs were cultured with na&x00EE;ve CD4+ T cells for 5 days. The production of cytokines was measured by intracellular staining and ELISA. Results Upon stimulation with TLR7 and TLR9 agonists, there were three main pDC populations: non-producers, TNF-α-producers, TNF-α/IFN-α-producers. Exogenous TNF-α significantly reduced the production of both IFN-α and TNF-α in TLR9-stimulated pDCs but only IFN-α in TLR7-stimulated pDCs. Neutralisation of autologous TNF-α with anti-TNF antibody partially sustained IFN-α secretion by TLR9-stimulated pDCs after 24 hours. Exogenous TNF-α significantly promoted pDC maturation by upregulation of costimulatory molecules and chemokine receptors such as CD80, CD86, HLA-DR, and CCR7. RNAseq data analysis suggested that TNF-α inhibits IFN-α/TNF-α production by interfering with the NFκB pathway, promotes antigen processing and presentation pathways as well as T cell activation and differentiation. Indeed, TNF-α-treated pDCs induced in vitro higher CD4+ T cell proliferation and favoured Th1/Th17 polarisation. Conclusions Although pDCs possess weak antigen-presenting properties, TNF-α can enhance pDC maturation by switching their main role as IFN-α-producing cells to a more conventional DC phenotype. The functional status of pDCs might be strongly influenced by overall inflammatory environment and TNF-α might regulate IFN-α-mediated aspects of a range of autoimmune and inflammatory diseases. Disclosure of Interest None declared

OP0095 Prediction of connective tissue disease in an at-risk cohort using a novel interferon stimulated gene expression score

Background A period of ANA positivity and other immune dysregulation precedes connective tissue disease, providing a potential opportunity for disease prevention. Type I interferons (IFN-I) play a role in pathogenesis but their role in disease initiation is unclear. Objectives To develop biomarkers of progression to systemic autoimmunity, with a view to enabling early intervention for disease prevention. Methods A prospective observational study was conducted in 125 patients At Risk of CTD defined by (i) ANA; (ii) ≤1 clinical SLE criteria; (iii) symptom duration <12 months and (iv) treatment-naïve. Progression was defined by meeting 2012 ACR/SLICC SLE, 2016 ACR/EULAR Primary Sjogrens, or other diagnostic criteria. Expression of 30 selected=“selected” ISGs was measured using Taqman. Factor analysis was used to reduce the gene expression data to a limited set of factors, which were compared between patient groups using Mann Whitney U Test. Two factor scores explained 80% of the data variance; “Score A” (composed of IFN-α responsive genes) and “Score B” (genes responsive to IFN-α and γ). 95 healthy controls and 107 SLE patients were used as negative and positive controls. Results 82 patients with 1-year follow-up data were studied. 71 were female with mean age 48±15 years. 16 (20%) patients progressed to CTD (SLE=12, Sjogrens=4) in the following 12 months. At baseline, only IFN Score A was increased in both At Risk-CTD and SLE vs healthy controls; p<0.001. IFN Score B was only increased in true established SLE. In At-Risk patients, IFN Score B was low in patients who did not progress and increased in those who did progress; p=0.004. However, there was no difference in IFN Score A between these two groups; p=0.252 (Figure 1). Although complement levels and lymphocyte counts were lower in SLE, these were not different between the At-Risk progression and non-progression groups. Anti-dsDNA titres were higher in SLE but not different between the progression groups; all p>0.10. Conclusions A novel ISG score predicts progression from ANA+ to clinical autoimmune disease. This may allow early intervention to prevent CTD. Analysis of other clinical, immunological and imaging biomarkers are in progress as well as a validation cohort. Disclosure of Interest M. Y. Md Yusof: None declared, Y. El-Sherbiny Grant/research support from: AstraZeneca, A. Psarras: None declared, E. Hensor: None declared, A. Alase: None declared, A. Mohamed: None declared, M. Wittmann: None declared, P. Emery Grant/research support from: AstraZeneca, E. Vital Grant/research support from: AstraZeneca

THU0272 Incomplete and Systemic Lupus Erythematosus Reveal A Different Pattern of Interferon-Stimulated Genes Up-Regulation

Background Type I interferons (IFN-I) play a central role in the pathogenesis systemic lupus erythematosus (SLE). IFN-I activity has been used as a biomarker by measuring interferon-stimulated gene (ISG) transcripts in the peripheral blood of SLE patients, but its relationship with clinical features still remains unclear. Incomplete lupus erythematosus (ILE) describes individuals with new onset of features suggestive of SLE, but do not fulfil diagnostic criteria. Up to 20% of these patients eventually progress to SLE. Objectives To investigate ISG expression in patients with ILE and explore possible qualitative differences between established and early stages of SLE. Methods A meta-analysis of known ISGs indicated 33 genes of importance [1]. The expression of 33 ISGs was measured using qPCR in PBMCs from individuals with SLE (n=54), ILE (n=27), and healthy controls (HC; n=14). SLE was defined using 2012 ACR/SLICC criteria. ILE was defined as ANA +ve, 1–2 clinical ACR/SLICC criteria, and symptom duration <12 months. Factor analysis (FA) was used to reduce expression data to a limited set of factors, which were compared between patient groups using ANCOVA test. Results FA on SLE patients indicated two factor scores explaining 80% of the data variance. The majority of variability was explained by Factor F1; however, Factor F2 appeared more relevant to the presence of fully established SLE. F1 and F2 were significantly different between patient groups; p=0.005 and p=0.044 respectively. F1 was similarly high in both SLE [SLE:HC=4.22 (1.80, 9.88), p=0.001] and ILE [ILE:HC=2.96 (1.20, 7.32), p=0.019]. In contrast, F2 was increased only in SLE [SLE:HC=1.38 (0.95, 2.00), p=0.086] but not in ILE [ILE:HC=1.02 (0.69, 1.51), p=0.917]; a significant difference was observed between SLE and ILE patients [SLE:ILE=1.35 (1.04, 1.77), p=0.026].In total, 16 and 14 genes loaded onto F1 and F2 respectively. Most of the genes were involved in IFN signalling pathways. Genes related to apoptosis and ubiquitination were present in both F1 and F2. Additionally, F1 loaded genes were mainly associated with antiviral immunity, complement regulation, and Th2 responses; genes loaded onto F2 were associated with regulation of IFN-I, dsDNA binding, chemotaxis, and Th1 responses. Conclusions IFN-I activity is present in ILE. However, the majority of measured ISG expression (F1) cannot distinguish ILE and SLE. F1 represents genes that distinguish healthy individuals, but show little variation at different stages of disease development. We define a subset of ISGs (F2), the expression of which is only increased in patients with confirmed clinical SLE. ISG expression is not unidimensional: qualitative differences in expression of distinct ISGs can contribute to clinical progression after disease initiation. References Chiche L et al, A&R 2014 Acknowledgement Dr Alaa Mohamed, Dr Ahmed Zayat and Dr Adewonuola Alase Disclosure of Interest None declared