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Objective JIA is an autoimmune, inflammatory disease with involvement of innate and adaptive immune responses. However, the role of neutrophils in JIA pathogenesis remains unclear. This study aimed to identify and validate neutrophil gene expression signatures in JIA using public microarray datasets and new clinical samples. Methods Three suitable datasets were analysed by significance analysis of microarray and Ingenuity. Neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from a new cohort of JIA patients and healthy paediatric controls (HCs). Gene expression was validated using quantitative PCR. Serum concentrations of proteins were measured using ELISA. Low-density granulocytes (LDGs) in JIA and HC PBMCs were quantified by flow cytometry using forward/side-scatter properties. Results Ingenuity identified transcriptional regulation (false discovery rate < 0.05) by G-CSF, GM-CSF and IL-8 along with expression of neutrophil granule protein genes including ELANE, MPO, MMP8 and MMP9 in datasets from JIA PBMCs. LDG counts were elevated in JIA compared with HCs (2.5% vs 1.4%; P = 0.007). Transcripts for MMP8 (P = 0.005), MPO (P = 0.0124) and Fcγ Receptor 1B (FCγR1B) (P = 0.0417) were significantly higher in JIA compared with HC neutrophils. MMP9 protein levels were lower in systemic JIA patient sera [355.95 ng/ml (s.d. 250.03)] compared with HCs [675.41 ng/ml (s.d. 181.17); P = 0.007], but levels of elastase, MPO and MMP8 were not significantly different. Conclusion LDGs are elevated in JIA and contribute to the transcriptomic profile of JIA PBMCs. JIA neutrophils express higher levels of MMP8 and FCGR1B, which may be implicated in disease pathology through the release of proteases and reactive oxygen metabolites, causing systemic inflammation and damage to joints.

Development of antiviral therapy against acute viral diseases, such as dengue virus (DENV), suffers from the narrow window of viral load detection in serum during onset and clearance of infection and fever. We explored a biomarker approach using 18F-fluorodeoxyglucose (18F-FDG) PET in established mouse models for primary and antibody-dependent enhancement infection with DENV. 18F-FDG uptake was most prominent in the intestines and correlated with increased virus load and proinflammatory cytokines. Furthermore, a significant temporal trend in 18F-FDG uptake was seen in intestines and selected tissues over the time course of infection. Notably, 18F-FDG uptake and visualization by PET robustly differentiated treatment-naive groups from drug-treated groups as well as nonlethal from lethal infections with a clinical strain of DENV2. Thus, 18F-FDG may serve as a novel DENV infection-associated inflammation biomarker for assessing treatment response during therapeutic intervention trials.

Background T cells are important to systemic lupus erythematosus (SLE) disease progression. This study determined the pro-inflammatory potential of T cells within the rare condition juvenile-onset SLE (JSLE). Method IL-17A and Th1/Th2-related cytokine concentrations were measured in plasma/serum from JSLE patients (n = 19, n = 11) and HC (n = 18, n = 7). IL17A, RORC, IL23 and IL23R mRNA were measured in peripheral blood mononuclear cells (PBMCs) from JSLE and healthy controls (HC) (n = 12). Th17-associated cytokine expression was analysed in the supernatant of CD3/CD28 activated JSLE (n = 7) and HC (n = 6) PBMCs. Results JSLE plasma IL-17A level (21.5 ± 5.2 pg/ml) was higher compared to HC (7.2 ± 2.5 pg/ml, p = 0.028). No differences were found in Th1/Th2 cytokines levels. IL = 17A (p = 0.022), IL-6 (p = 0.028) and IL-21 (p = 0.003) concentrations were increased in supernatants from activated JSLE PBMCs. IL-17 F (p = 0.50) and IL-22 (p = 0.43) were also increased but were not statistically significant. IL17A and IL23 mRNA was significantly higher in JSLE PBMCs (p = 0.018 and p = 0.01). Conclusion JSLE T cells have an increased ability to secrete Th17 associated cytokines once activated, which could contribute to the pro-inflammatory disease phenotype seen in these patients.

Background Neutrophils are one of the first cell types to be recruited to sites of inflammation or infections, yet have been implicated in a wide range of non-infectious inflammatory conditions. Low-density granulocytes (LDGs) are a distinct subset of neutrophils recently documented in a number of systemic autoimmune diseases such as Systemic Lupus Erythematosus (SLE) (1). LDGs show increased ability to undergo neutrophil extracellular trap (NET) formation, leading to potential autoantigen presentation, and subsequent inflammation (2,3). Citrullination of histones in neutrophils, a post-translational histone modification, is a critical step in NET formation (4). Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. We have previously shown increased LDG expression in JSLE patients; however histone citrullination in JSLE and JIA, and LDG populations in JIA has yet to be characterized. Objectives To compare LDG expression in JIA peripheral venous blood to healthy controls. To investigate the presence of histone citrullination in JSLE and JIA patients compared to healthy controls. Methods While mature neutrophils typically sediment with the red-blood cell fraction in a Ficoll-density gradient, LDGs co-purify in the PMBC fraction. LDGs and neutrophils were isolated from whole blood using HetaSep and magnetic bead separation. The percentage populations of LDGs were analysed using flow cytometry in JIA and healthy paediatric controls. Where possible, protein was extracted from the neutrophils of each patient group. Citrullinated histone 3 relative to the expression of histone 3 was measured using Western Blot analysis in JIA and JSLE patients compared to controls. Results are presented as mean ± SEM. Results Preliminary data suggests the expression of LDGs in the peripheral blood of JSLE patients (n=11; 8.31%±2.5) and JIA patients (n=3; 3.6%±2.4) is increased compared to healthy paediatric controls (n=4; 1.62%±0.95). Overall, the ratio of citrullination of histone 3 to pan histone 3 was higher in JSLE neutrophils (0.75±0.15) compared to healthy controls (0.38±0.12; p Conclusions Increased expression of LDGs was observed in JIA and JSLE patients compared to controls. This supports previous research from our group that has shown increased expression of LDGs in JSLE patients correlates with disease activity. LDGs may therefore play an integral role in triggering an autoimmune inflammatory response. Notably, citrullination of histone 3 was increased in JSLE neutrophils compared to controls, but not JIA neutrophils. This may indicate that JSLE neutrophils are intrinsically more primed to undergo NETosis compared to JIA patients. Further investigations are being carried out to increase patient size and to characterize LDG phenotype and function. References Carmona-Rivera C, Kaplan MJ. Semin Immunopathol, 2013. 35(4): p. 455-63 Villanueva E, et al. The Journal of Immunology 2011 July 01;187(1):538-552. Denny MF, et al. The Journal of Immunology 2010 March 15;184(6):3284-3297. Wang, Y, et al. J Cell Biol, 2009. 184(2): p. 205-13 Disclosure of Interest None declared

Background Abnormal cytokine expression is postulated to be a factor in the immunopathology of Systemic Lupus Erythematosus (SLE), leading to inflammation and organ damage. Several studies have shown elevated levels of IL-6, IL-17, IL-21 and TNF-α in adult SLE serum and plasma using ELISAs 1,2 . A spike-and-recovery assessment is used to measure the analyte recovery difference between a natural sample matrix and the standard diluent. Generally 80-120% is considered an acceptable range of recovery. A previous spike-and-recovery study of SLE serum and plasma has shown poor recovery of high mobility group box 1 (HMGB1) protein 3 . Objectives To validate and assess the assay response of juvenile-onset SLE (JSLE) serum and plasma in IL-6, IL-17, IL-21 and TNF-α detection kits. Methods JSLE plasma and serum samples were assayed in spike-and-recovery and linearity-dilution experiments. Three spike-and-recovery ELISA assessments with IL-21 (Biolegend), IL-6 and TNF-α (R&D Systems) were performed, each including 3 JSLE plasma samples and 1 control sample. IL-17 spike-and-recovery ELISA (eBioscience) was executed with 2 JSLE serum and 2 JSLE plasma samples. A spike-and-recovery assessment with IL-17 was performed on 5 JSLE serum samples using a single Bio-Plex Assay (Bio-Rad). Percentage recovery of respective cytokines in spiked and unspiked samples were then calculated and compared against the concentration of spiked sample diluents. Results Recovery percentages in JSLE samples were less than 80% in TNF-α, IL-17 and IL-21 assays (Fig. 1), apart from IL-17 and TNF-α at 1 in 8 dilution which yielded 80% and 113% respectively. Measuring IL-21 in JSLE and control samples showed poor recovery out of acceptable range at all 4 dilutions. IL-6 concentrations fell within the acceptable range and demonstrated good responses in JSLE and control samples. Conclusions Interference was observed in the assay response of JSLE serum and plasma in IL-17, IL-21 and TNF-α assays but not IL-6 ELISA. Recovery increased with sample matrix dilution, indicating that dilution reduced the interference, however this must be balanced against the risk of diluting out the analyte. It can be inferred that constituents in the sample matrix may be affecting the detection of these cytokines. JSLE plasma/serum cytokine concentrations measured by ELISA and single plex assays must be interpreted with caution. Methods such as using specialist buffers or alternative assays should be considered to minimise interference and to increase robustness of future studies. Spike-recovery experiments using a multiplex assay platform are currently underway. References Ohl K et al. J Biomed Biotechnol. 2011;2011. Davas EM et al. Clin Rheumatol. 1999;18(1):17-22. Urbonaviciute V et al. J Leukoc Biol. 2007;81(1):67-74. Disclosure of Interest None Declared