Background/Purpose: Most children with juvenile dermatomyositis (JDM) have chronic disease despite multi-drug immunosuppression, highlighting the need for targeted therapies. Neutrophil extracellular traps (NETs) are key in autoimmunity, but their role in JDM is unclear. This study examined neutrophil activation and NET formation, focusing on calprotectin as a candidate biomarker and mediator of tissue damage.

Methods: Plasma calprotectin was measured in JDM patients (n=51) and healthy controls (n=10). Sixteen endothelial cell analytes were assessed in 48 JDM patients. Proteomic profiling of six treatment-naïve samples was done using the Olink 3072 panel. Ex vivo and in vitro assays tested JDM plasma and recombinant calprotectin on muscle cell injury. Anti-NXP2 antibodies from treatment-naïve patients were evaluated for their ability to induce NETs by SYTOX Green-based assay.

Results: Circulating calprotectin levels were significantly elevated in JDM patients compared to healthy controls (p < 0.0001). Calprotectin strongly correlated with absolute neutrophil counts (r = 0.63, p < 0.0001), consistent with its origin from activated neutrophils and its link to NET formation. Notably, higher calprotectin levels were also associated with established biomarkers of muscle injury, including creatine phosphokinase (r = 0.34, p = 0.036), aldolase (r = 0.40, p = 0.013), and lactate dehydrogenase (r = 0.37, p = 0.023). Clinically, elevated calprotectin reflected reduced muscle strength, as assessed by lower Manual Muscle Testing-8 scores (r = –0.44, p = 0.007), and higher Physician’s Global Activity Assessment scores (r = 0.36, p = 0.023), underscoring its potential as a biomarker of disease activity. Furthermore, calprotectin levels showed strong correlations with multiple soluble EC analytes—including Ang-2, Tie2, Gal-9, and sTNFR-II—suggesting a role in endothelial activation or injury. Plasma proteomic expression profiling in TN JDM patients also revealed significant upregulation of multiple neutrophil activation proteins (AZU1, MPO, S100A11, S100A12) (Fig. 1). Mechanistic studies demonstrated that calprotectin-rich JDM plasma induced marked cytotoxicity and microcalcification in cultured human skeletal muscle cells (Fig. 2 A-G). These effects were recapitulated by recombinant calprotectin, confirming a direct pathogenic role. Additionally, anti-NXP2 antibodies purified from JDM plasma robustly induced NET formation when incubated with neutrophils from healthy donors, implicating this JDM-specific autoantibody in neutrophil activation and NETosis (Fig. 3 A-B). Ongoing experiments aim to assess the potential direct pathogenic effects of anti-NXP2 on endothelial cells and muscle tissue.

Conclusion: Calprotectin is a mechanism-informed biomarker in JDM that reflects neutrophil activation and correlates with muscle disease activity, endothelial dysfunction, and muscle damage. Anti-NXP2 autoantibodies trigger NETosis, linking adaptive immunity to neutrophil-driven pathology. These findings reveal a novel pathogenic axis at the intersection of innate and adaptive immunity with vascular health, suggesting new targets for biomarker-guided therapy.

Figure 1. Upregulation of neutrophil activation and NET formation protein expression signature in JDM. Heatmap displaying individual-level Z-score Normalized Protein Expression (NPX) values of select neutrophil-specific proteins in treatment-naïve (TN) JDM plasma (n = 6) compared to healthy controls (n = 9). These proteins were also significantly associated with global disease activity, highlighting a neutrophil-driven inflammatory signature at the time of JDM diagnosis.

Figure 2. Impact of elevated calprotectin—both from high-calprotectin JDM samples and recombinant protein—on cytotoxicity and calcification in human skeletal muscle myoblasts (HSMMs). (A) Schematic of the skeletal muscle cell cytotoxicity assay. (B) Cytotoxicity of HSMMs was assessed via LDH release after 24-hour treatment with 2.5% plasma from JDM subjects with high (n=10) or low (n=10) circulating calprotectin. (C) Correlation between plasma calprotectin levels and LDH release in JDM samples. (D) LDH release from HSMMs following treatment with recombinant calprotectin (20µg/mL). (E) Alizarin Red staining of HSMMs treated with recombinant calprotectin (10 µg/mL) in calcification medium (PI) for 6 days, with media refreshed every 2–3 days. (F) Calcium content was quantified in parallel wells; calcium deposits were dissolved with 1N HCl for 3 hours at 4°C and measured using the QuantiChrom Calcium Assay Kit. (G) Schematic of intracellular calcium accumulation assay. *Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. The JDM-specific autoantibody anti-NXP2 IgG induces NETosis in healthy donor neutrophils. (A) Schematic of the SYTOX Green-based NETosis assay. (B) Healthy donor neutrophils were treated with anti-NXP2 IgG (100 µg/mL) for 6 hours in the presence of SYTOX Green, which labels extracellular DNA released during NETosis. *Significance: **P < 0.01.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/clinical-and-mechanistic-insight-of-circulating-calprotectin-and-nxp2-autoantibodies-in-juvenile-dermatomyositis/