Hyaluronan-mediated modulation of human neutrophil function

Hyaluronan (HA) is a glycosaminoglycan that in its natural, high molecular mass (HMM) form, promotes tissue repair and homeostasis. With inflammation, enhanced HA metabolism results in HMM HA fragmentation to low molecular mass (LMM) fragments that may act as a damage-associated molecular pattern to initiate innate immune responses. However, the responsiveness of myeloid cells to HA is controversial and largely unknown for neutrophils. Here, I investigated if and how HA can directly affect key immune functions of neutrophils and potentially contribute to the dysregulated neutrophil activation observed in childhood-onset rheumatic diseases. For this investigation, peripheral blood cells from healthy donors were incubated ex vivo with pharmaceutical grade HA of different molecular mass (HMM, LMM and HA fragments < 10 kDa) alone and in the presence of a pro-inflammatory cytokine, TNF. Key neutrophil functions, namely cytokine production, reactive oxygen species release (ROS), granule mobilization and apoptosis were assessed. HA alone had no effect on neutrophil cytokine production, granule mobilization and apoptosis. However, HA primed neutrophils for a robust release of extracellular ROS in response to secondary stimuli. An effect that was further enhanced when HA and TNF were added together. Unlike described mechanisms of priming by classical stimuli such as TNF, HA-mediated priming and co-priming (with TNF) were independent of granule mobilization yet, correlated with the activation (phosphorylation) of p38 MAPK. Additionally, HA enhanced TNF-induced baseline production of intracellular ROS. This coincided with an increased rate of apoptosis in HA/TNF stimulated cells compared to TNF alone. Concomitantly, I have demonstrated that circulating concentrations of HA were elevated in children with systemic lupus erythematosus and tracked with disease activity. Thus, I designed an experimental panel to detect primed neutrophils in a small quantity of whole blood, suitable for clinical samples. Together, this study provides new evidence that hyaluronan is a novel neutrophil priming agent, which enhances ROS production to secondary stimuli. Moreover, it describes the function of HA as a modulator of TNF-induced neutrophil function. My study suggests a previously unrecognized contribution of HA to neutrophil functionality and potentially the inflammatory response that involves extensive neutrophil infiltration and turnover of ECM components.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Comparable type I interferon score determination from PAXgene and Tempus whole blood RNA collection and isolation systems

Objective: Type I interferons (IFN) have important roles in many immune-mediated inflammatory diseases (IMIDs) and are a relatively new therapeutic target. Direct detection of type I IFNs has proved challenging, thus their presence is often inferred from the expression of interferon-stimulated genes (ISGs) and calculation of an interferon score (IS). The objective of this research was to determine if the expression of six common ISGs and subsequent IS were comparable when RNA was derived from the Tempus and PAXgene whole blood RNA collection systems. Results: Whole blood was obtained from ten healthy adults, incubated ex vivo in the absence and presence of recombinant human IFNα then divided into PAXgene and Tempus tubes. Despite reports of tube-specific patterns of gene expression, quantitative PCR (qPCR) analysis revealed no significant differences between PAXgene and Tempus tubes in either the homeostatic or IFNα-induced expression of six ISGs (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1). Overall there was a strong correlation in the IS between unstimulated (r = 0.92, p = 0.0005) and IFNα-stimulated (r = 0.71, p = 0.0268) samples derived from the PAXgene and Tempus tubes.Medicine, Faculty ofScience, Faculty ofOther UBCMedicine, Department ofMicrobiology and Immunology, Department ofPediatrics, Department ofRheumatology, Division ofReviewedFacult

Periodic fever syndromes: beyond the single gene paradigm

Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease in Canada and is characterized by a clinical syndrome of episodic inflammatory symptoms. Traditionally, the disease is defined by autosomal recessive inheritance of MEFV gene variants, yet FMF also not uncommonly manifests in individuals with only one identified disease-associated allele. Increasing availability and affordability of gene sequencing has led to the identification of multiple MEFV variants; however, they are often of unknown clinical significance. Variants in other genes affecting overlapping or distinct inflammatory signaling pathways – together with gene-environment interactions including epigenetic modulation – likely underlie the significant genetic and phenotypic heterogeneity seen among patients with this disease. We review recent evidence of the expanding spectrum of FMF genotype and phenotype and suggest that current drug funding schemes restricting biologic agents to patients with homozygous mutations have not kept pace with our biological understanding of the disease.Medicine, Faculty ofScience, Faculty ofMedicine, Department ofMicrobiology and Immunology, Department ofRheumatology, Division ofReviewedFacult

Complexity in unclassified auto-inflammatory disease: a case report illustrating the potential for disease arising from the allelic burden of multiple variants

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.Medicine, Faculty ofScience, Faculty ofOther UBCNon UBCMedical Genetics, Department ofMicrobiology and Immunology, Department ofPediatrics, Department ofReviewedFacult