Hyaluronan promotes intracellular ROS production and apoptosis in TNFα-stimulated neutrophils.

Background: Hyaluronan (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. Methods: Blood 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. Results: Hyaluronan 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. Conclusions: These 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.

Children with systemic autoinflammatory diseases have multiple, mixed ethnicities that reflect regional ethnic diversity.

OBJECTIVES: To evaluate the ethnic diversity of children with a systemic autoinflammatory disease (SAID) in a multi-ethnic Canadian province. METHODS: Self-reported ethnicity of 149 children and adolescents with a SAID in British Columbia, Canada, was analysed for ethnic representation among individual patients, across the cohort, within particular SAIDs, and compared to provincial census data on ethnic diversity. RESULTS: Half of reported cases had a diagnosis of either PFAPA (23.5%) or an unclassifiable autoinflammatory syndrome (31.5%), with a monogenic SAID diagnosed in only 12.8% of cases. The majority of participants (73.1%) were mixed ethnicity with European and Asian heritage reported most frequently (57.0% and 23.0% of all responses, respectively). Ethnic diversity reflected regional diversity except for West Asian, Arabic, Jewish, and Eastern European heritage, which were over-represented in SAID patients, and Chinese descent, which was under-represented in our cohort compared to the general population of British Columbia. CONCLUSIONS: Results from this study show extensive multi-ethnic diversity in individual patients and across the various SAIDs inclusive of monogenic SAIDs that are frequently associated with particular ethnicities. Although not disproportionately represented, this is the first report of systemic autoinflammatory disease in Canadian children of Indigenous heritage.

Hyaluronan primes the oxidative burst in human neutrophils.

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 <10 kDa). Key innate immune functions were assessed, namely production of cytokines and reactive oxygen species release (ROS), granule mobilization, and apoptosis. None of the tested sizes of HA altered cytokine production by PBMC and neutrophils. Also, HA had no effect on neutrophil granule mobilization and apoptosis. In contrast, HA primed neutrophils for rapid and robust release of ROS in response to a secondary stimulus (N-formyl-methionyl-leucyl phenylalanine). Priming occurred within 20 min of exposure to HA and was similar for all tested molecular mass. The observed effect was independent of granule mobilization and associated with the activation of intracellular signaling pathways involving Src family kinases, glycogen synthase kinase-3, and the proline-rich Akt substrate of 40 kDa. Our findings provide new evidence that HA, irrespective of molecular mass, is a specific priming agent of the neutrophil oxidative burst, which is a critical, early component of an innate immune response.

Anti-neutrophil cytoplasmic antibodies (ANCA): Antigen interactions and downstream effects.

Neutrophils are the most abundant leukocytes in circulation and are key "first responders" in the immune response to infectious and non-infectious stimuli. Unlike other immune cells, neutrophils can mount a robust response (including a change in surface markers and the production of extracellular traps and reactive oxygen species) just minutes after sensing a disturbance. It has been speculated that, in some individuals, the activation of neutrophils inadvertently leads to the generation of anti-neutrophil cytoplasmic autoantibodies (ANCA) against particular neutrophil proteins (antigens) such as myeloperoxidase (MPO) and proteinase 3 (PR3). In these individuals, continuous ANCA-antigen interactions are thought to drive persistent activation of neutrophils, chronic immune activation, and disease, most notably, small vessel vasculitis. There are significant gaps however in our understanding of the underlying mechanisms and even the pathogenicity of ANCA given that vasculitis can develop in the absence of ANCA, and that ANCA have been found in circulation in other conditions with no apparent contribution to disease. These gaps are particularly evident in the context of human studies. Herein, we review knowledge on neutrophil-derived ANCA antigens PR3 and MPO, ANCA generation, and ANCA-antigen interaction(s) that may promote immune activation and disease.

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.

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.

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.

Methods for type I interferon detection and their relevance for clinical utility and improved understanding of rheumatic diseases.

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.