Background/Purpose: Neutrophils are key immune cells participating in host defense through several mechanisms, including the formation of neutrophil extracellular traps (NETs). Although beneficial from a host-pathogen perspective, exaggerated NET formation has been linked to inflammation and autoimmunity, including lupus and adult dermatomyositis (DM). However, NETs have not been described in juvenile DM or pediatric overlap syndromes, such as RNP+ myositis. One debilitating manifestation of chronic JDM is calcinosis, the formation of calcium deposits/crystals in soft tissue. Though other crystals, including cholesterol and monosodium urate crystals are known activators of neutrophils, the role of calcium deposits/crystals in neutrophil activation and subsequent cell death is not known. In the current study, we aim to investigate if calcium crystals could mediate NETosis in vitro, and to determine the clinical relevance of calcium crystal-mediated NETosis in children with Juvenile Myositis.

Methods:

Neutrophils were activated with synthesized calcium crystals and analyzed for NET formation in presence of reactive oxygen species (ROS) inhibitors using immunofluorescence microscopy and ELISA. Markers of neutrophil activation (S100A8/A9, peroxidase activity) and NETs (MPO-DNA complexes) were analyzed in plasma from healthy children (HC, n=20), pediatric lupus (n=10), polymyositis (n=7), JDM patients (n=66), and RNP+ myositis (9). In the Juvenile Myositis population, 74% were female, with a mean age of 11.3 years. The association of NETs with clinical parameters was tested, including disease activity scores (DAS) for skin, muscle and total, the presence of calcinosis as well as Myositis Specific Antibodies (MSA) and Myositis Associated Antibodies (MAA).

Results:

Levels of S100A8/A9 and peroxidase activity were markedly elevated in JDM patients as a group (p<0.01), as well as in children with RNP+ antibodies (p=0.005), indicating systemic neutrophil activation. In children with RNP autoantibodies, S100A8/A9 levels correlated with total disease activity score (r=0.64, p<0.05) as well as disease activity score in the skin (r=0.80, p<0.001). No associations were found between NETs and MSA. Consistent with our hypothesis, calcium crystals induced neutrophil activation in vitro, with up-regulation of the adhesion molecules CD66b and CD11b on the neutrophil cell surface (p<0.05). Further, calcium crystals induced prominent NETosis (p<0.05) in a ROS-dependent manner, as determined by fluorimetry and immunofluorescence microscopy. Children with calcinosis had increased levels of circulating NETs as compared to calcinosis-negative children (p=0.003) as well as healthy controls (p=0.02).

Conclusion: Our results demonstrate a clear contribution of neutrophils in JM pathogenesis, and suggest calcium crystal-driven NETosis is a novel, and potentially therapeutically targetable, mechanism participating in calcinosis-associated pathology. Thus, monitoring neutrophil-derived markers may have significant clinical utility in patients with Juvenile Myositis and skin involvement.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/calcium-crystal-mediated-netosis-in-juvenile-dermatomyositis-and-anti-rnp-overlap-syndrome-with-skin-involvement/