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BackgroundHyaluronan (HA) is an important structural component of the extracellular matrix and has well-described roles in maintaining tissue integrity and homeostasis. With inflammation, HA metabolism (synthesis and degradation) increases and results in higher concentrations of soluble HA. Previously, we demonstrated that (soluble) HA primed resting neutrophils for the oxidative burst in response to a secondary stimulus. Notably, HA-mediated priming was not dependent on degranulation, which is a hallmark of priming by classical agents such as TNFα. In this study, we queried the ability of HA to prime neutrophils to different stimuli and its capacity to modulate neutrophil function in the presence of TNFα.MethodsBlood neutrophils from healthy donors were stimulated ex vivo with HA in the absence and presence of classic neutrophil agonists, inclusive of TNFα. Western blotting was used to assess the activation (phosphorylation) of p38 MAPK, and key neutrophil functions associated with priming and activation, such as intracellular and extracellular ROS production, degranulation, and apoptosis, were evaluated by standard chemiluminescence assays (ROS) and flow cytometry.ResultsHyaluronan is capable of atypical priming and, with TNFα, co-priming neutrophils for an enhanced (rate and/or magnitude) oxidative burst to various secondary stimuli. In addition, HA can augment intracellular ROS production that is directly induced by TNFα in resting neutrophils, which coincided with the activation of p38 MAPK and apoptosis.ConclusionsThese data demonstrate that the extracellular matrix component HA is a key modulator of neutrophil function(s) in the presence of inflammatory agents such as TNFα. Moreover, it provides additional evidence for the diversity and complexity of neutrophil priming and activation during inflammation.

Hyaluronan (HA) is a glycosaminoglycan that in its natural, high molecular mass (HMM) form, promotes tissue repair and homeostasis. With inflammation, HA metabolism and HMM HA fragmentation to low molecular mass (LMM) forms is greatly enhanced. Considerable evidence suggests that LMM HA may act as a damage-associated molecular pattern to initiate innate immune responses. However, the responsiveness of myeloid cells to LMM HA is controversial and largely unknown for neutrophils. Peripheral blood cells from healthy donors were incubated ex vivo with pharmaceutical grade HA of different molecular mass (HMM, LMM, and HA fragments

Background: Type I interferons (IFN) have important roles in many pediatric and adult rheumatic diseases and are a new therapeutic target for which several “anti-interferon (anti-IFN)” treatments are currently in use or in development. Since the direct detection of these proteins in biological samples has proved challenging, indirect methods are often used to infer the presence of type I IFN. Most commonly this involves quantification of the relative expression of interferon-stimulated genes (ISGs) that are used to calculate an interferon score (IS) (1). This score has been used for example to asses type I IFN activity in pediatric patients with type I interferonopathies, systemic lupus erythematosus, dermatomyositis and systemic juvenile idiopathic arthritis (2). Both qPCR and Nanostring technology have similar sensitivity and reproducibility for IS determination (3). The use of different whole blood RNA collection systems on the IS have not been evaluated however despite evidence of method-dependent changes in gene expression (4). Objectives: The aim of the study was to compare expression of six common ISGs (IFI27, IFI44L, IFIT1, ISIG15, RSAD2, SIGLEC1) and the corresponding IS in RNA derived from two commonly used whole blood RNA collection systems (PAXgene and Tempus). Methods: Whole blood was collected from ten healthy individuals (median age 25.5 years) in sodium heparin tubes and incubated without or with recombinant human interferon alpha 2b (rhIFNα, 2 IU/ml, 4 hrs, 37C°, 5% CO2). Next, samples were divided between PAXgene (PreAnalytiX, Becton Dickinson) and Tempus (Applied Biosystems) tubes and RNA was isolated according to the manufacturer’s protocols. cDNA was synthesized (∼500ng input RNA; qScript cDNA synthesis kit) and ISG expression measured on a QuantStudio 6 Real-Time PCR instrument using a TaqMan Fast Advanced Assay. For each ISG, expression was normalized against the geometric mean of two housekeeping genes (18s rRNA and HPRT1) and calculated using the formula 2-ΔCt. Relative gene expression is reported as the normalized expression of each ISG divided by the median of normalized expression of the same ISG in unstimulated samples. The median relative expression of all six ISGs was used to calculate the IFN score for each sample. Results: There was no statistically significant difference in the normalized expression of any of the six ISGs in either the rhIFNα-stimulated or unstimulated samples derived from PAXgene or Tempus tubes. The greatest difference in mean normalized expression in both unstimulated and stimulated samples was observed for ISG15 (difference in mean normalized expression was 0.0034 and 0.11, respectively). Overall there was a strong correlation of the IFN score between PAXgene and Tempus tubes for both the unstimulated (R2 = 0.9117, p Conclusion: Despite reported differences in gene expression patterns associated with samples collected in PAXgene versus Tempus tubes, our results demonstrate that 6-gene interferon scores do not differ significantly between RNA samples obtained with these two systems. These results suggest that health care and research centres can use either tubes for IFN score determination using these 6 ISGs and results can be directly compared irrelevant of the RNA collection system employed. References: [1] Rice GI et al, Lancet Neurol 2013; 12: 1159-69; [2] Rice GI et al, J Clin Immunol 2017; 37: 123-32 [3] Pescarmona R et al, Cytokine. 2019;113:446-452.; [4] Skogholt AH et al, BMC Research Notes 2017; 10:136. Disclosure of Interests: None declared

Background Despite recent advances in the diagnosis and understanding of many autoinflammatory diseases, there are still a great number of patients with phenotypes that do not fit any clinically- and/or genetically-defined disorders. Case presentation We describe a fourteen-year-old boy who presented at two and a half years of age with recurrent febrile episodes. Over the course of the disease, the episodes increased in frequency and severity, with new signs and symptoms continuing to appear. Most importantly, these included skin changes, splenomegaly and transaminitis. Only partial control of the disease was achieved with anti-IL-1 therapy. Extensive investigation showed generalized inflammation without immune deficiency, with increased levels of serum amyloid A and several pro-inflammatory cytokines including interferon-γ, as well as an increased type I interferon score. Exome sequence analysis identified P369S and R408Q variants in the MEFV innate immunity regulator, pyrin (MEFV) gene and T260 M and T320 M variants in the NLR family pyrin domain containing 12 (NLRP12) gene. Conclusion Patients with unclassified and/or unexplained autoinflammatory syndromes present diagnostic and therapeutic challenges and collectively form a substantial part of every cohort of patients with autoinflammatory diseases. Therefore, it is important to acquire their full genomic profile through whole exome and/or genome sequencing and present their cases to a broader audience, to facilitate characterization of similar patients. A critical mass of well-characterized cases will lead to improved diagnosis and informed treatment.

Type I interferons (IFN) are a class of inducible and protective cytokines best known for immune defence against viruses and intracellular bacteria. Inappropriate stimulation or defective negative regulation of type I IFN expression however can lead to persistent type I IFN activity with detrimental effects. This is particularly relevant for a class of monogenic autoinflammatory diseases ("type I interferonopathies"), along with many other complex rheumatic diseases such as systemic lupus erythematosus (SLE), dermatomyositis (DM), systemic sclerosis (SSc), rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Direct detection of type I interferon protein in biologic samples has proved challenging, thus indirect methods are often used to infer the presence of type I IFN via quantification of antiviral activity and/or induced expression of IFN-responsive genes. While some of these methods have been used to inform clinical care, none have proven feasible for everyday clinical practice. However, with new technologies emerging, this may soon change. This review provides a brief summary of the available methods to gauge the presence of type I IFN and their application for the improved understanding, diagnosis and monitoring of type I interferonopathies and other rheumatic diseases.